Human Kallikrein Research Articles

Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI.

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191-Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4-BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

Ligand screening assay for the enzyme kallikrein immobilized on NHS-activated Sepharose

Human tissue kallikreins (KLKs) are serine proteases involved in various physiological and pathological conditions, including cancer and neurological disorders. These enzymes constitute attractive drug targets, which has stimulated the search for new KLK inhibitors. In this study, we have covalently immobilized porcine pancreas KLK on an NHS-activated Sepharose matrix, to obtain KLK-Sepharose-NHS. The immobilized enzyme showed high recovered activity and maintained the ability of free KLK to recognize the synthetic substrate Z-Phe-Arg-AMC (KMapp = 10.3 ± 0.9 μM). As proof of concept, we used leupeptin as a reference inhibitor to perform inhibition studies for KLK-Sepharose-NHS and to determine the half-maximal inhibitory concentration (IC50 = 0.13 ± 0.01 μM), the inhibition constant (Ki = 0.06 μM), and the leupeptin inhibition mechanism. We evaluated several complex matrixes (plant crude extract) by the same bioassay, to demonstrate their applicability. The species Solanum lycocarpum, Stryphnodendron adstringens, and Psychotria carthagenensis gave the best results. KLK-Sepharose-NHS was fully active after six consecutive reaction cycles and retained about 60 % of its initial activity after being used for at least five months, so the bioassay developed herein is a promising strategy to screen and to identify KLK ligands.

Pretherapeutic first-in-human targeting trial of anti-hK2 antibody h11B6: A potential molecular vector for alpha-particle radioimmunotherapy.

122 Background: Radioligand therapy for metastatic castration resistant prostate cancer (mCRPC) has focused to date on targeting prostate specific membrane antigen, an approach associated with salivary gland toxicity and variable lesion expression. New targets are needed. Human kallikrein-2 (hK2) is overexpressed in most prostate cancer, with minimal normal tissue expression or serum secretion. Humanized monoclonal antibody (mAb) h11B6 exclusively targets hK2 in pre-clinical studies. This phase 0 study (NCT04116164) is a first-in-human trial of [111In]-DOTA-h11B6 to determine the hk2-targeting potential of h11B6. Objective: The purpose of this ongoing protocol is to characterize the biodistribution of [111In]-DOTA-h11B6 and determine a suitable mAb mass that targets mCRPC with minimal non-tumor targeting. Methods: Patients with progressive mCRPC were enrolled. A single slow bolus infusion of 2 mg [111In]-DOTA-h11B6 (nominally 185 MBq [111In]), was administered IV with or without 8 mg h11B6. Patients were observed for adverse events (AE) for at least 2 weeks. Serial gamma camera imaging including at least one SPECT/CT scan was performed up to 8 days post-administration. Serial blood samples were obtained over 2 weeks to determine serum radioactivity and h11B6 protein levels. Dosimetry for normal organs was estimated using OLINDA-EXM. Results: Results for the first 6 patients are summarized in Table. Treatment was tolerated in all patients with no AEs and no evidence of enhanced accumulation in any organ including salivary glands. Initial volume of distribution appeared confined to the vascular compartment. Slow clearance of radioactivity from the vascular compartment was observed with gradual targeting to skeletal and non-skeletal lesions in all patients. Conclusions: h11B6 is a novel mAb that targets hK2, localizes to bone and soft tissue metastases, has no significant normal tissue uptake, and spares salivary glands. An IND for a phase I therapeutic study using [225Ac] has been secured. Clinical trial information: NCT04116164. [Table: see text]

Pharmacological and clinical study results of Berotralstat Hydrochloride for long-term prophylactic treatment of hereditary angioedema

Hereditary angioedema (HAE) is a rare disease that causes serious health problem and affects on quality of life for patient due to recurrent episodes of angioedema in various body such as the skin, larynx, digestive tract, and limbs. Many HAE patients have deficiency or dysfunction of C1 inhibitor, impaired regulation of plasma kallikrein activity and overproduction of bradykinin, resulting in leading to episodes of increased capillary hyper permeability and angioedema. Therapy of HAE consists of on-demand treatment for acute attack and prophylactic treatment by suppressing the onset of acute attack in the short and long term. However, no drug has been approved for long-term prophylaxis in Japan. Berotralstat hydrochloride (ORLADEYO Capsules 150 mg) is an oral, selective plasma kallikrein inhibitor approved for the suppression of the onset of acute attacks in HAE in Japan in January 2021. Preclinical studies demonstrated that Berotralstat is a potent and highly specific inhibitor of human plasma kallikrein activity. Berotralstat suppressed bradykinin production in the HUVEC system. Clinical studies demonstrated that oral administration of Berotralstat to HAE type I or type II patients at a dose of 150 mg once daily showed a reduction of HAE attack rate and clinically significant change in angioedema quality of life score. The most common side effect was gastrointestinal symptoms. In conclusion, preclinical and clinical data indicated that Berotralstat is an effective treatment for long-term prophylactic treatment by suppressing the onset of acute attack in HAE patient and is considered to be a useful treatment option for patients.

Human Tissue Kallikrein 1 Is Downregulated in Elderly Human Prostates and Possesses Potential In Vitro Antioxidative and Antifibrotic Effects in Rodent Prostates.

Objective The aim of the present study was to investigate the protective effects and mechanisms of KLK1 on aging-related prostate alterations and search clues about the application of KLK1 to the treatment of human BPH. Methods Thirty-six rats including 26 male wild-type SD rats and 10 transgenic rats were fed to 3- or 18-month-old and divided into three groups: young WTR (yWTR) as the control (n = 16), aged WTR (aWTR) (n = 10), and aged TGR (aTGR) (n = 10). The prostates of the three groups of rats (10 rats per group) were harvested to evaluate the levels of KLK1 expression, oxidative stress, fibrosis, and involved signaling pathways, such as NO/cGMP, COX-2/PTGIS/cAMP, and TGF-β1/RhoA/ROCK1, via quantitative PCR, Western blot, histological examinations, and ELISA. Moreover, the remaining 6 yWTRs were sacrificed to obtain primary prostate fibroblast and aortic endothelial cells, and a coculture system was built with the cells for the verification of above signaling pathways in vitro. And the direct effects of bradykinin on prostate cells were detected by MTT experiment. Prostate specimens of 47 patients (age from 48 to 92 years) undergoing BPH surgery were collected after approval. Histological examinations and KLK1 IHC were preformed to analyze the relationship between KLK1 expression and age and prostate fibrosis. Results The human KLK1 gene only existed and was expressed in aTGR. The prostate of young rats expressed more KLK1 than the aged and the expression of KLK1 in prostate decreased with age in humans (r = −0.347, P = 0.018). Compared to the aWTR group, the yWTR and aTGR groups showed milder fibrosis, less oxidative stress, upregulated NO/cGMP, and COX-2/PTGIS/cAMP signaling pathways and inhibited TGF-β1/RhoA/ROCK1 signaling pathway. In the coculture system, KLK1 suppressed TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation via cleaving LMWK to produce the BK which upregulate eNOS expression and NO production in endothelial cells. BK not only slightly stimulated the proliferation ability of prostatic stromal cells but also upregulated iNOS and inhibited TGF-β1 expression in them. Conclusion KLK1 protects prostate from oxidative stress and fibrosis via amplified NO/cGMP signal in aged rats. The decrease of KLK1 expression with aging is laying the groundwork for the application of KLK1 to the treatment of human BPH. The current experimental data showed that the side effects of KLK1 on the prostate cell were not obvious.

Structural modelling and thermostability of a serine protease inhibitor belonging to the Kunitz-BPTI family from the Rhipicephalus microplus tick

rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.

Co‑expression of tissue kallikrein1 and tissue inhibitor of matrix metalloproteinase1 improves myocardial ischemia‑reperfusion injury by promoting angiogenesis and inhibiting oxidative stress.

Myocardial ischemia/reperfusion (I/R) injury is a serious complication of reperfusion therapy for myocardial infarction. At present, there is not an effective treatment strategy available for myocardial I/R. The present study aimed to investigate the effects of human tissue kallikrein 1 (hTK1) and human tissue inhibitors of matrix metalloproteinase 1 (hTIMP1) gene co-expression on myocardial I/R injury. A rat model of myocardial I/R injury and a cell model with hypoxia/reoxygenation (H/R) treatment in cardiac microvascular endothelial cells (CMVECs) were established, and treated with adenovirus (Ad)-hTK1/hTIMP1. Following which, histological and triphenyl-tetrazolium-chloride staining assays were performed. Cardiac function was tested by echocardiographic measurement. The serum levels of oxidative stress biomarkers in rats and the intracellular reactive oxygen species (ROS) levels in CMVECs were measured. Additionally, experiments, including immunostaining, reverse transcription-quantitative PCR, western blotting, and MTT, wound healing, Transwell and tube formation assays were also performed. The results of the present study demonstrated that Ad-hTK1/hTIMP1 alleviated myocardial injury and improved cardiac function in myocardial I/R model rats. Ad-hTK1/hTIMP1 also significantly enhanced microvessel formation, decreased matrix metalloproteinase (MMP)2 and MMP9 expression, and reduced oxidative stress in myocardial I/R model rats. Furthermore, Ad-hTK1/hTIMP1 significantly enhanced proliferation, migration and tube formation in H/R-treated CMVECs. Additionally, Ad-hTK1/hTIMP1 significantly decreased intracellular ROS production and γ-H2A.X variant histone expression levels in H/R-treated CMVECs. In conclusion, the results of the present study demonstrated that co-expression of hTK1 and hTIMP1 genes displayed significant protective effects on myocardial I/R injury by promoting angiogenesis and suppressing oxidative stress; therefore, co-expression of hTK1 and hTIMP1 may serve as a potential therapeutic strategy for myocardial I/R injury.

Generation of recombinant antibodies against human tissue kallikrein 7 to treat skin diseases

Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.

Ahp-Cyclodepsipeptides as tunable inhibitors of human neutrophil elastase and kallikrein 7: Total synthesis of tutuilamide A, serine protease selectivity profile and comparison with lyngbyastatin 7

We describe the total synthesis of tutuilamide A, a potent porcine pancreatic elastase (PPE) inhibitor and a representative member of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide family, isolated from marine cyanobacteria. The Ahp unit serves as a pharmacophore and the adjacent 2-amino-2-butenoic acid (Abu) is a main driver of the selectivity among serine proteases. We adapted our previous convergent strategy to generate the macrocycle, common with lyngbyastatin 7 and related elastase inhibitors, and then appended the tutuilamide A-specific side chain bearing a vinyl chloride. Tutuilamide A and lyngbyastatin 7 were evaluated side by side for the inhibition of the disease-relevant human neutrophil elastase (HNE). Tutuilamide A and lyngbyastatin 7 were approximately equipotent against HNE, while tutuilamide A was previously shown to be more active against PPE compared with lyngbyastatin 7, further demonstrating that the side chain provides opportunities to not only modulate potency but also selectivity among proteases of the same function from different organisms. Profiling of tutuilamide A against mainly human serine proteases revealed high selectivity for HNE (IC50 0.73 nM) and pleiotropic activity against kallikrein 7 (KLK7, IC50 5.0 nM), without affecting other kallikreins, similarly to lyngbyastatin 7 (IC50 0.85 nM for HNE and 3.1 nM for KLK7). A comprehensive molecular docking study for elastases and KLK7 afforded deeper insight into the intricate differences between inhibitor interactions with HNE and PPE, accounting for the differential activities for both compounds. The synthesis and molecular studies serve as a proof-of-concept that the macrocyclic scaffold can be diversified to fine-tune the activity of serine protease inhibitors.

Human Tissue Kallikreins in Polymorphous Adenocarcinoma: A Polymerase Chain Reaction and Immunohistochemical Study.

Polymorphous adenocarcinoma (PAC) is the second most common malignant salivary gland tumour of minor salivary glands. Human tissue kallikreins (KLKs) are a family of highly conserved serine proteases expressed by various tissues and organs. The literature demonstrates a link between KLKs and salivary gland neoplasms. The purpose of this study was to determine levels of KLK mRNA in tissue samples of PAC and to determine if KLK expression is limited to tumour cells. Nineteen cases of PAC were reviewed (1987-2013). The diagnosis was confirmed, demographic data was collected, and formalin fixed paraffin-embedded PAC and normal salivary gland tissue samples were obtained. RNA isolation was achieved, followed by conversion to complementary DNA via reverse transcription. Using PCR, the quantitative level of expression of KLKs1-15 was recorded. Samples exhibiting high and low KLK expression were selected for immunohistochemistry staining. Results revealed a statistically significant increase in mean KLK mRNA expression for KLK1, KLK4, KLK10, KLK12 and KLK15 in PAC tissue samples, compared with normal salivary gland tissue (Mann-Whitney U test, p < 0.05). Immunohistochemistry results demonstrated that KLKs were present in tumor cells. Notably, all samples demonstrating relatively higher KLK mRNA expression showed equivalent or increased staining scores relative to the low KLK mRNA expression samples. In conclusion, there appears to be aberrant kallikrein expression in polymorphous adenocarcinoma, suggesting the possibility of a kallikrein cascade influence on tumor development and progression.

Structural Studies on the Inhibitory Binding Mode of Aromatic Coumarinic Esters to Human Kallikrein-Related Peptidase 7.

The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small-molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding, His57 undergoes an outward rotation; thus, the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.

Aseminalkiller ou quand un animal de compagnie vous asphyxie…

Patients and methodsHerein we report the case of a 60-year-old female patient who for a number of months had presented angioedema of the airways repeatedly after sexual intercourse. The allergological investigations militated in favour of an allergy to seminal fluid with specific IgE directed against seminal fluid positive at 1.55 kIU/L. Dog IgE was positive and Can f 5 (canine prostatic kallikrein) was positive at 63 kIU/L. Questioning revealed that the patient had acquired a new dog a year earlier. Her new dog is a male bichon whereas before she had a female dog. Use of condoms prevented further recurrence of angioedema. DiscussionCanine prostatic kallikrein (Can f 5) shares cross-antigenicity with human prostatic kallikrein (PSA protein). This 28-kDa molecule is present in dog urine and dander. Cross-antigenicity sets have been described. Hypersensitivity can occur from the first episode of sexual intercourse as a result of prior sensitisation to canine kallikrein. ConclusionCertain allergies to seminal fluid are linked to cross-antigenicity between canine prostatic kallikrein (Can f 5) and human prostatic kallikrein. The originality of our case lies in the description of an acquired form of allergy to seminal fluid, whose link with the recent acquisition of a male dog appears relevant.

Human Tissue Kallikrein 1 Improves Erectile Dysfunction of Streptozotocin-Induced Diabetic Rats by Inhibition of Excessive Oxidative Stress and Activation of the PI3K/AKT/eNOS Pathway

Objective To investigate the protective effects and mechanisms of human tissue kallikrein 1 (hKLK1) on type 1 diabetes mellitus- (DM-) induced erectile dysfunction in rats. Materials and Methods. The homozygous transgenic rats (TGR) harboring the hKLK1 gene and age-matched wild-type Sprague Dawley rats (WTR) were involved, and intraperitoneal injection of streptozotocin was utilized to induce diabetes in rats. Forty-eight-week-old male rats were randomly divided into a WTR group, TGR group, diabetic WTR group (WTDM), diabetic TGR group (TGDM), and TGDM with HOE140 group (TGDMH), with eight rats in each group. Twelve weeks later, the erectile response of all rats was detected by cavernous nerve electric stimulation, and corpus cavernosums were harvested to evaluate the levels of cavernous oxidative stress (OS), apoptosis, fibrosis, and involved pathways. Moreover, cavernous smooth muscle cells (CSMC) and endothelial cells (EC) were primarily isolated to build a coculture system for a series of in vitro verification. Results The hKLK1 gene and age-matched wild-type Sprague Dawley rats (WTR) were involved, and intraperitoneal injection of streptozotocin was utilized to induce diabetes in rats. Forty-eight-week-old male rats were randomly divided into a WTR group, TGR group, diabetic WTR group (WTDM), diabetic TGR group (TGDM), and TGDM with HOE140 group (TGDMH), with eight rats in each group. Twelve weeks later, the erectile response of all rats was detected by cavernous nerve electric stimulation, and corpus cavernosums were harvested to evaluate the levels of cavernous oxidative stress (OS), apoptosis, fibrosis, and involved pathways. Moreover, cavernous smooth muscle cells (CSMC) and endothelial cells (EC) were primarily isolated to build a coculture system for a series of Conclusions hKLK1 preserves erectile function of DM rats through its antitissue excessive OS, apoptosis, and fibrosis effects, as well as activation of the PI3K/AKT/eNOS/cGMP pathway in the penis. Moreover, hKLK1 promotes relaxation and prevents high glucose-induced injuries of CSMC mediated by EC-CSMC crosstalk.

Phase 0 study to assess [111In]-DOTA-h11B6 to target human kallikrein-2 (hk2) in men with metastatic castration-resistant prostate cancer.

TPS249 Background: Human kallikrein-2 (hk2) is a member of the kallikrein family, is produced by prostatic epithelium, has 80% DNA sequence homology with PSA, is both circulating and bound to prostatic tissue, and is highly expressed even in high grade tumors. hK2 holds the promise of serving as a new therapeutic target for radiopharmaceuticals in prostate cancer. h11B6 is a humanized antibody directed to an epitope found on the catalytically active form of hK2, which is permanently internalized after binding. This is a first-in-human trial of [111In]-DOTA-h11B6 to determine the radio-immunotherapeutic potential of targeting hK2 with nanomolar affinity. The trial will determine the most favorable antibody mass as well as assess tumor targeting. Methods: To determine the optimal antibody mass, two cohorts of patients will be studied. All patients will receive 2 mg of [111In]-labeled h11B6. The first cohort consists of escalating amounts of antibody mass in 2-6 patients: 2, 10 and 20 mg total antibody respectively. Patients will undergo serial whole-body images for up to a week, along with assessments of serum clearance kinetics and normal organ residence time. The lowest mass amount of antibody that demonstrates favorable biodistribution and evidence of radioactivity accumulation in tumor will be used for the second cohort. Up to 6 patients will be studied in the second cohort, which will focus on assessment of antibody tumor targeting. Following this optimization and targeting imaging trial, a phase 1 dose-escalation radioimmunotherapy trial may be performed. Clinical trial information: NCT04116164.

NEW MONOCLONAL ANTIBODIES TO THE PROSTATE-SPECIFIC ANTIGEN: OBTAINING AND STUDYING BIOLOGICAL PROPERTIES

Original set of 15 clones of hybridomas producers of monoclonal antibodies (mAbs) to prostate-specific antigen (PSA) (using as a source of lymphocytes of mice of inbred strains NZB and Balb/c) was obtained. The activity in ELISA, affinity constant and titer in the culture fluid of the mAbs obtained from NZB mice are higher compared to the mAbs obtained from the Balb/c mice splenocytes. Conjugates of obtained mAbs with horseradish peroxidase were synthesized, and it allowed performing comparative epitope characterization of the resulting set of mAbs. Studied mAbs are directed to 4 epitopes of PSA molecule: 2 mAbs interacting with one of the epitopes showed cross-activity with PSA-related protein – human kallikrein 2; 2 mAbs of the same epitope specificity blocked the enzymatic activity of PSA; mAbs of the remaining two epitopes either did not affect PSA chymotrypsin activity at all, or this inhibitory effect was not significant. The obtained mAbs recognize antigenic determinants that are not screened when interacting with α1-antichymotrypsin, as well as those that are localized in the places where α1-antichymotrypsin interacts with PSA. Within each of the given two mAbs groups, there are antibodies belonging to different epitopes, which give prospects for the further use of the obtained mAbs in immunoassay and immunobiotechnology. Two alternative methods for determining of the mAbs constants of affinity (by Friguet and Scatchard) are comparable (for determination of the affinity constants of anti-PSA antibodies): coefficient of linear correlation between the affinity constants defined by different methods was 0.90.

Plasma Kallikrein Contributes to Ellagic Acid-Induced Coagulation in the Absence of FXI By Activating Factor IX

Background - Factor XI (FXI) plays a critical role in thrombosis, as its inhibition reduces clot formation in animal models, and human congenital FXI deficiency appears to protect against venous thromboembolism and stroke. In contrast, in both settings, the impact on hemostasis is rather low, especially compared to FIX deficiency. Little is known about the cause of this difference. As thrombin generation can be observed in FXI-deficient plasma, we speculated that intrinsic activation of FIX is not solely dependent on FXI(a). Aim - Evaluate whether plasma kallikrein contributes to FIX activation and subsequent thrombin generation in FXI deficiency. Methods - Kaolin-initiated coagulation and fibrin formation was assessed in different plasma types in the presence or absence of an active site inhibitor of human plasma kallikrein (PKa) using the activated partial thromboplastin time (aPTT). Thrombin generation was measured in ellagic acid-induced contact activation in human FXI-deficient plasma in the presence or absence of the PKa inhibitor. Levels of FIXa-antithrombin (FIXa-AT) or FXa-AT were determined at defined time points using in-house developed ELISAs. The role of thrombin and its substrate FVIII in ellagic acid-induced contact activation in the absence of FXI was investigated using dabigatran or a human monoclonal FVIII antibody and assessing the FIXa-AT and FXa-AT plasma levels. In in vivo experiments, F11-/- mice were treated with ellagic acid in the presence or absence of the PKa inhibitor and FXIa-AT levels were assessed 30 min after administration. Results - Plasma kallikrein inhibition dose-dependently prolonged kaolin-triggered clotting time in human FXI-deficient plasma but not in normal pooled or plasma prekallikrein depleted plasma, indicating a role for PKa in coagulation in the absence of FXI. In FXI-deficient plasma, ellagic acid activated the coagulation system, as could be demonstrated by measurement of thrombin generation, and FIXa-AT and FXa-AT levels, which immediately reached the upper detection limit of 1.6 nM suggesting a FXI bypass route towards FIX activation. Addition of a PKa inhibitor caused a delayed and diminished FXa-AT and FIXa-AT complex formation and led to a reduction in thrombin peak height (EC50: 3 µM). This indicates that PKa might contribute to FXI-independent coagulation by activating FIX. In the presence of dabigatran FIXa-AT complex formation in FXI-deficient plasma was completely inhibited, which could be reversed by addition of purified thrombin. In the presence of purified thrombin, a FVIII antibody inhibited thrombin-dependent FIXa-AT complex formation suggesting that the activation of FVIII by thrombin is essential for FIX activation by PKa or to support the activity of FIXa. In line with this, thrombin generation in FXI-defcient plasma was inhibited by the FVIII antibody. In ellagic acid-treated F11-/- mice, FIXa-AT complex formation was significantly increased compared to controls (612 pM vs. 121 pM; P &lt; 0.001), and was attenuated upon inhibition of PKa. Conclusions - We demonstrate that, activation of FXII leads to thrombin generation via FIX activation by plasma kallikrein in the absence of FXI. In addition, the activation of FVIII by thrombin is essential for the FXI bypass route towards FIX activation. These findings support the idea that FXI and FIX have distinctly different roles within the intrinsic coagulation cascade upon FXII activation and may contribute to variations in bleeding in FXI-deficient subjects due to different prekallikrein plasma levels. Disclosures Visser: Bayer AG: Employment. Laux:Bayer AG: Employment. Heitmeier:Bayer AG: Employment.

Discovery of novel plasma biomarker ratios to discriminate traumatic brain injury

Background: Traumatic brain injury (TBI) is a major cause of death and disability. Despite increased awareness, reliable biomarkers are urgently needed to aid in all forms of traumatic brain injury diagnosis and prognosis. Methods: Here, we aim to assess the diagnostic utility of known and novel TBI biomarkers in a pilot patient cohort of severe TBI (sTBI) patients and healthy controls. We analyzed concentrations of S100 calcium binding protein B (S100B), neuron specific enolase (NSE), human kallikrein 6 (hK6) and prostaglandin D2 synthase (PGDS) using ELISA immunoassays. Results: Plasma levels of hK6 and PGDS were significantly lower in sTBI compared with controls, while S100B and NSE were significantly higher. Furthermore, we show that ratios of NSE and S100B with hK6 and PGDS may be able to determine the presence of sTBI better than single markers alone. Conclusions: The findings presented here represent a starting point for future validation, where biomarker ratios can be tested in independent TBI cohorts.

M1 muscarinic receptor is a key target of neuroprotection, neuroregeneration and memory recovery by i-Extract from Withania somnifera

Memory loss is one of the most tragic symptoms of Alzheimer’s disease. Our laboratory has recently demonstrated that ‘i-Extract’ of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders.

Discovery of novel plasma biomarker ratios to discriminate traumatic brain injury.

Background: Traumatic brain injury (TBI) is a major cause of death and disability. Despite increased awareness, reliable biomarkers are urgently needed to aid in all forms of traumatic brain injury diagnosis and prognosis. Methods: Here, we aim to assess the diagnostic utility of known and novel TBI biomarkers in a pilot patient cohort of severe TBI (sTBI) patients and healthy controls. We analyzed concentrations of S100 calcium binding protein B (S100B), neuron specific enolase (NSE), human kallikrein 6 (hK6) and prostaglandin D2 synthase (PGDS) using ELISA immunoassays. Results: Plasma levels of hK6 and PGDS were significantly lower in sTBI compared with controls, while S100B and NSE were significantly higher. Furthermore, we show that ratios of NSE and S100B with hK6 and PGDS may be able to determine the presence of sTBI better than single markers alone. Conclusions: The findings presented here represent a starting point for future validation, where biomarker ratios can be tested in independent TBI cohorts.

The Effect of Human Urinary Kallikrein on Corticospinal Tract Injury in Acute Ischemia Stroke Patients

The Effect of Human Urinary Kallikrein on Corticospinal Tract Injury in Acute Ischemia Stroke Patients