High Molecular Weight Kininogen: A Review of the Structural Literature

Kininogens are multidomain glycoproteins found in the blood of most vertebrates. High molecular weight kininogen demonstrate both carrier and co-factor activity as part of the intrinsic pathway of coagulation, leading to thrombin generation. Kininogens are the source of the vasoactive nonapeptide bradykinin. To date, attempts to crystallize kininogen have failed, and very little is known about the shape of kininogen at an atomic level. New advancements in the field of cryo-electron microscopy (cryoEM) have enabled researchers to crack the structure of proteins that has been refractory to traditional crystallography techniques. High molecular weight kininogen is a good candidate for structural investigation by cryoEM. The goal of this review is to summarize the findings of kininogen structural studies.

Cleavage of High Molecular Weight Kininogen and Bradykinin Release By Red Blood Cell Microvesicles As a Putative Mechanism for Hypotensive Transfusion Reactions

Background: Hypotensive transfusion reactions are adverse events typified by a sudden decrease in blood pressure that usually occurs within the first minutes after the initiation of a transfusion and resolves shortly after the transfusion is stopped. Due to current passive reporting practices, the incidence is likely underreported, but recent studies estimate an incidence of 1.3 cases per 10000 RBC units. The pathophysiology of these reactions are not fully understood. One hypothesis proposed is that increased bradykinin (BK), a nonapeptide released from cleavage of high molecular weight kininogen (HK), as seen with the use of negatively charged leukoreduction filters and the use of angiotensin-converting enzyme inhibitors, is a major contributor to the pathophysiology. We have recently demonstrated that red blood cell derived microvesicles (RBCMVs) from aging red blood cell (RBC) units are able to trigger thrombin generation via kallikrein activation - a predominant enzyme to cleave high molecular weight kininogen (Noubouossie, Blood, 2020). Thus, we hypothesize that the same RBCMVs would lead to bradykinin generation and might explain these hypotensive events. Objectives: To determine if RBC storage lesion-derived microvesicles are able to facilitate HK cleavage and BK release. Methods: RBCMVs were prepared from 4 recently expired RBC units (42 or 43 day old, AS-3 preserved, prestorage leukoreduced, all A+) via a series of centrifugations and washes. RBCMVs were quantified and characterized using nanoparticle tracking analysis. Obtained RBCMVs were first assessed for the capacity to initiate thrombin generation in microvesicle free human plasma via a substrate cleavage assay. Next, RBCMVs were added to a buffer reaction containing prekallikrein and HK, and kininogen cleavage was assessed via western blot. RBCMVs were also mixed with microvesicle-free human plasma and analyzed for evidence of kallikrein activation, cleavage of high molecular weight kininogen, and bradykinin production by ELISA. Cohn fractionation of plasma was used to enrich for BK. Results: RBCMVs were enumerated and concentrated to 7.5 ± 1.4 x 10 12 per mL (mean±SD size 160 ± 29µm). RBCMVs were able to initiate thrombin generation principally via contact pathway activation, independently of tissue factor. In a buffer system RBCMVs demonstrated activity to generate kallikrein with a sequential high molecular weight kininogen cleavage in a dose-dependent manner. Exclusion of kallikrein from the buffer system or addition of the small molecule inhibitor of kallikrein - ecallantide - halted cleavage of kininogen. A dose-dependent cleavage of high molecular weight kininogen indicated that RBCMVs could cause BK release in plasma; this was confirmed via an independent assay of Cohn -fractionated samples. Conclusions: Results of this current study demonstrate that RBCMVs are leading to high HK cleavage via kallikrein activation in vitro. We suspect that the same mechanisms could lead to BK generation in patients receiving older RBC units, possibly increasing the risk for hypotensive events from transfusion. Disclosures Karafin: Westat, Inc.: Consultancy. Key: Sanofi: Consultancy; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator.

Immune Checkpoint Inhibitors (ICI) Enhance Cancer Associated Thrombosis (CAT) in Murine Model of Colon Carcinoma

Abstract Background: Venous thromboembolism (VTE) is an important immune-related adverse event (irAE) associated with immune checkpoint inhibitors (ICI) cancer therapy. To better understand the pathogenesis of ICI-induced VTE during ICI treatment, we utilized a murine model of cancer associated thrombosis (CAT) to examine the impact of ICI treatment on the development of flow restriction-induced thrombosis. Methods A syngeneic colon carcinoma murine model (CT26) on BALB/c background was utilized to evaluate venous thrombosis following inferior vena cava (IVC) ligation. Non-tumor-bearing and tumor-bearing animals were treated with therapeutic doses of ICI: anti-programmed cell death receptor 1 antibody (anti-PD1) and cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4) or isotype control antibodies. Mice underwent surgical treatment for IVC ligation followed by surgical retrieval of thrombus. Western blot analysis was performed on plasma cleaved high molecular weight kininogen (cHK), and citrullinated histone H3 (CitH3). Results Tumor-bearing mice undergoing IVC ligation after anti-PD1 and anti-CTLA4 infusion developed larger thrombi compared to mice treated with isotype control IgG. Thrombus weights in CT26 tumor-bearing mice treated with ICI (19.56±4.41 mg) were significantly increased compared to those in mice treated with isotype control IgG (14.67±2.76 mg) (P=0.043). The weight of thrombi in non-tumor-bearing mice was not affected by ICI treatment (9.25±2.22 mg with control IgG vs 9.33±1.15 mg with ICI). In addition, there was a significant increase in thrombus length in mice treated with ICI (9±1.02 mm vs 7.61±0.99 mm with IgG, P=0.039). Cleaved high molecular weight kininogen (cHK), an indicator of contact activation was increased in plasma pre-IVC occlusion from ICI-treated mice compared to IgG-treated mice (68% vs 38% in HK cleavage). Citrullinated histone H3 (CitH3), a NETosis marker, was elevated in plasma (and in thrombus) post-IVC occlusion from ICI-treated mice compared to that of IgG-treated mice. Conclusions ICI treatment of tumor-bearing mice undergoing flow restriction-induced thrombosis resulted in enhanced clot formation. Larger thrombi in ICI treated mice was accompanied with enhanced contact activation and NETosis marker CitH3. Figure 1 Figure 1. Disclosures Khorana: Halozyme: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Anthos: Consultancy, Honoraria. McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

Severe High Molecular Weight Kininogen (HK) Deficiency: Clinical Characteristics, Deficiency-Causing KNG1 Variants in Reported and New Cases, and Estimated Prevalence

Background: Severe high molecular weight kininogen (HK) deficiency is an autosomal recessive defect of the contact system caused by mutations in KNG1. Limited scientific interest in HK deficiency due to the rarity of the seemingly asymptomatic condition may increase, as HK, the precursor of bradykinin, is now discussed as a therapeutic target e.g. in hereditary angioedema. Aims: We provide a comprehensive analysis of the diagnostic, clinical, and genetic features of HK deficiency and estimate its frequency. Methods: We identified a new case of HK deficiency, systematically review the literature, conduct new genetic studies of reported cases, and comprehensively analyze the clinical course and diagnostic criteria. Clotting activity of HK and prekallikrein (PK) (HK:C/PK:C) and antigen (HK:Ag/PK:Ag) were determined and genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. Characteristics deduced from the known HK deficiency-causing variants were used to estimate the frequency of HK deficiency from the KNG1 variants aggregated in GnomAD. Results: 677 studies were identified by systematic review of the literature for HK deficiency. 27 of these contained individual cases of HK deficiency including 6 studies not listed in PubMed. Little-noticed cases from the gray literature account for more than one-third (16/39) of the extracted, unrelated cases. We genotyped one newly diagnosed HK-deficient case and 2 cases described in the literature and additionally evaluated all 10 studies reporting genetic data in HK-deficiency (including one case previously misdiagnosed as having PK deficiency). A total of 10 KNG1 variants causing HK deficiency (one new) were found, the most frequent being c.586C>T, p.Arg196* (4 unrelated families). Interestingly, all HK deficiency-causing variants are truncating, whereas two amino acid substitutions with presumed functional consequence, have been described as the cause of hereditary angioedema. Conservative prevalence estimates based on all known and putative HK deficiency-causing variants extracted from GnomAD (truncating variants in KNG1, including indels, nonsense and canonical splice site mutations located in that part of the gene, where relevant mutations have been described) revealed a frequency of 1 case of HK deficiency among 7,925,172 with slight differences in the analyzed ethnicities (see table). In addition, although not to the same extent as seen in PK deficiency, HK deficiency apparently is more prevalent in Africans. While it is already well known that HK deficiency causes decreased PK levels, our data indicate that factor XI levels are also frequently decreased, albeit to a lesser extent. The number of cases detected so far is too low for a more detailed analysis regarding bleeding, thrombotic, and cardiovascular events or immunological abnormalities. Conclusion: HK-deficiency is probably more frequent than previously thought. Suspected cases of contact phase defects should at least be analyzed for HK activity (besides factor XII, XI and PK activity) to facilitate conclusive evaluation of the clinical significance in the future. Figure 1 Figure 1. Disclosures Lämmle: Takeda: Membership on an entity's Board of Directors or advisory committees; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Baxter: Other: Travel Support, Speakers Bureau; Alexion: Other: Travel Support, Speakers Bureau; Siemens: Other: Travel Support, Speakers Bureau; Bayer: Other: Travel Support, Speakers Bureau; Roche: Other: Travel Support, Speakers Bureau; Sanofi: Other: Travel Support, Speakers Bureau.

Plasmin-mediated Cleavage of High Molecular Weight Kininogen Contributes to Acetaminophen-Induced Acute Liver Failure

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (FXII and PK) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high molecular weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, ELISA, and mass spectrometry analysis demonstrated that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic, but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Hereditary angioedema: Investigational therapies and future research

The future therapies for hereditary angioedema will likely involve the development of oral agents as alternatives to parenteral administration of drugs, specific targeting of proteins and/or enzymes that are not yet possible (e.g., factor XIIa), new agents that target the β2 receptor with sustained action properties, testing of products to determine whether the β1 receptor contributes significantly to attacks of angioedema, disrupting protein synthesis by using RNA technology as an alternative to enzyme inhibition, and, finally, gene therapy to attempt to cure the disease. Complete inhibition of attacks may well require sustained blood levels of C1 inhibitor that exceed 85% of normal, and it may be possible to delete the prekallikrein gene (analogous to familial prekallikrein deficiency), which is the one factor that might alleviate bradykinin formation, even by factor XII‐independent initiating mechanisms, with the possible exception of Mannose Associated Serine Protease 1 (MASP-1) cleavage of high molecular weight kininogen (HK). Deletion of the light chain of high-molecular-weight kininogen would eliminate all possibilities for bradykinin formation, except tissue kallikrein cleavage of low-molecular-weight kininogen to support normal physiologic function to at least 50%.