WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency

Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.

Comparison of Contaminant Profiles and Biochemical Properties for Commercial Preparations of Immune Globulin Intravenous (IGIV)

Immune globulin intravenous (IGIV) has grown steadily in the volume of use and numbers of clinical indications since the first modern commercial preparation was approved by FDA in 1981. All US-marketed IGIV products are approved by FDA to treat primary immune deficiency; seven are also approved for immune thrombocytopenia purpura. Currently, most IGIV infusions are for treating autoimmune diseases. IGIV has historically been isolated from pools of human plasma (at least 1,000 donors, but typically ranging from 10,000 donations in the US up to 60,000 donations in some EU nations) using Cohn-Oncley cold ethanol fractionation. Most firms now supplement this process with chromatographic steps to remove Factor XIa, other promoters of complement activation, and other Ig isotypes. Despite high purity, a range of adverse symptoms, varying widely by product, is associated with IGIV infusions. To date, a possible correlation between adverse event profiles and contaminant profiles for IGIV products has not been studied and remains poorly understood. FDA requires donor blood tests for absence of hepatitis, HIV and other viral diseases. Final container immunoglobulin products must show potency against measles, diphtheria and polio virus and contain no prekallikrein activator. However, no further characterization of contaminant proteins is currently required. We therefore undertook comparative component analyses of 5% and 10% solution IGIV products approved or in development for the US market. Component analysis was performed using high resolution mass spectrometry (HRMS) and very sensitive multiplexed ELISA (mELISA) surveys to identify and quantitate trace contaminants in individual products. Factor XIa antigen and activity tests as well as functional assays were performed to assess the thrombogenic potential of the products. Subgroup and Ig isotype distribution were assessed by ELISA, and size exclusion chromatography was used to determine the extent of molecular aggregation. HRMS and mELISA are complementary but fundamentally different survey methods. HRMS is less "biased" in that it will detect any molecular entity more abundant than the limit of detection, whereas mELISA detected primarily biomarkers included in lists of assays preset by the vendor. Among these were several tumor markers, a wide range of inflammatory modulators, and a number of metabolic hormones. For a complex biologic such as polyclonal IGIV, HRMS typically detects entities present at levels of 10 - 20 molar ppm, while the lower limit of quantitation (LLOQ) of the mELISA is typically 3 - 5 orders of magnitude lower. Therefore, the mELISA survey of each IGIV product typically detects a substantially greater number of contaminant entities than the HRMS assay. If mELISA found a protein at ≥ 10 ppm for a product, it was generally also among the contaminants detected by HRMS. Thus, HRMS and mELISA results correlated satisfactorily. With both methods, detected levels of any given contaminant varied up to several orders of magnitude among the IGIV products tested. The most abundant contaminants in most preparations were IgA, IgD, β 2-glycoprotein-1, α 2-macroglobulin and albumin. Residual albumin and IgM were found in some but not all products. Inflammatory modulators were largely absent from the IGIV products surveyed. The interleukins IL6, IL-8 and IL-10 were detected in a few products, but in no case more than 4-fold above the mELISA LLOQ. The tumor markers carcinoembryonic antigen (CEA), α-fetoprotein (αFP), CA-125, CA 19-9 and tumor necrosis factor-α were not found in any product. The results of these assays are discussed with respect to their potential relationship to the observed adverse event and safety profiles of the individual products. Disclosures Abramson: GC Mogam, Incorporated: Consultancy, Research Funding. Van Cott: GC Mogam, Incorporated: Research Funding.

DAKS1, a Kunitz Scaffold Peptide from the Venom Gland of Deinagkistrodon acutus Prevents Carotid-Artery and Middle-Cerebral-Artery Thrombosis via Targeting Factor XIa

Scaffold-based peptides (SBPs) are fragments of large proteins that are characterized by potent bioactivity, high thermostability, and low immunogenicity. Some SBPs have been approved by the FDA for human use. In the present study, we developed SBPs from the venom gland of Deinagkistrodon acutus (D. acutus) by combining transcriptome sequencing and Pfam annotation. To that end, 10 Kunitz peptides were discovered from the venom gland of D. acutus, and most of which peptides exhibited Factor XIa (FXIa) inhibitory activity. One of those, DAKS1, exhibiting strongest inhibitory activity against FXIa, was further evaluated for its anticoagulant and antithrombotic activity. DAKS1 prolonged twofold APTT at a concentration of 15 μM in vitro. DAKS1 potently inhibited thrombosis in a ferric chloride-induced carotid-artery injury model in mice at a dose of 1.3 mg/kg. Furthermore, DAKS1 prevented stroke in a transient middle cerebral-artery occlusion (tMCAO) model in mice at a dose of 2.6 mg/kg. Additionally, DAKS1 did not show significant bleeding risk at a dose of 6.5 mg/kg. Together, our results indicated that DAKS1 is a promising candidate for drug development for the treatment of thrombosis and stroke disorders.