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.

Plasma Kallikrein Contributes to Coagulation in the Absence of Factor XI by Activating Factor IX.

OBJECTIVES: FXIa (factor XIa) induces clot formation, and human congenital FXI deficiency protects against venous thromboembolism and stroke. In contrast, the role of FXI in hemostasis is rather small, especially compared with FIX deficiency. Little is known about the cause of the difference in phenotypes associated with FIX deficiency and FXI deficiency. We speculated that activation of FIX via the intrinsic coagulation is not solely dependent on FXI(a; activated FXI) and aimed at identifying an FXI-independent FIX activation pathway. Approach and Results: We observed that ellagic acid and long-chain polyphosphates activated the coagulation system in FXI-deficient plasma, as could be demonstrated by measurement of thrombin generation, FIXa-AT (antithrombin), and FXa-AT complex levels, suggesting an FXI bypass route of FIX activation. Addition of a specific PKa (plasma kallikrein) inhibitor to FXI-deficient plasma decreased thrombin generation, prolonged activated partial thromboplastin time, and diminished FIXa-AT and FXa-AT complex formation, indicating that PKa plays a role in the FXI bypass route of FIX activation. In addition, FIXa-AT complex formation was significantly increased in F11-/- mice treated with ellagic acid or long-chain polyphosphates compared with controls and this increase was significantly reduced by inhibition of PKa. CONCLUSIONS: We demonstrated that activation of FXII leads to thrombin generation via FIX activation by PKa in the absence of FXI. These findings may, in part, explain the different phenotypes associated with FIX and FXI deficiencies.

Decreased plasma kallikrein activity is associated with reduced kidney function in individuals with type 1 diabetes.

AIMS/HYPOTHESIS: Plasma kallikrein is the central mediator of the plasma kallikrein-kinin system, which is involved both in vascular control and thrombin formation cascades. The plasma kallikrein-kinin system has also been considered protective in pathological conditions, but the impact of plasma kallikreins on diabetic nephropathy remains unknown. The objective of this cross-sectional study was to explore the association of plasma kallikrein with diabetic nephropathy. METHODS: We measured plasma kallikrein activity in 295 individuals with type 1 diabetes at various stages of diabetic nephropathy, and we tested the genetic association between the plasma kallikrein-kinin system and kidney function in 4400 individuals with type 1 diabetes. RESULTS: Plasma kallikrein activity was associated with diabetes duration (p < 0.001) and eGFR (p < 0.001), and plasma kallikrein activity was lower with more advanced diabetic nephropathy, being lowest in individuals on dialysis. The minor alleles of the KNG1 rs5030062 and rs710446 variants, which have previously been associated with increased plasma pre-kallikrein and/or factor XI (FXI) protein levels, were associated with higher eGFR (rs5030062 ß = 0.03, p = 0.01; rs710446 ß = 0.03, p = 0.005) in the FinnDiane cohort of 4400 individuals with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Plasma kallikrein activity and genetic variants known to increase the plasma kallikrein level are associated with higher eGFR in individuals with type 1 diabetes, suggesting that plasma kallikrein might have a protective effect in diabetic nephropathy.

Structures of full-length plasma kallikrein bound to highly specific inhibitors describe a new mode of targeted inhibition.

Plasma kallikrein (pKal) is a serine protease responsible for cleaving high-molecular-weight kininogen to produce the pro-inflammatory peptide, bradykinin. Unregulated pKal activity can lead to hereditary angioedema (HAE) following excess bradykinin release. HAE attacks can lead to a compromised airway that can be life threatening. As there are limited agents for prophylaxis of HAE attacks, there is a high unmet need for a therapeutic agent for regulating pKal with a high degree of specificity. Here we present crystal structures of both full-length and the protease domain of pKal, bound to two very distinct classes of small-molecule inhibitors: compound 1, and BCX4161. Both inhibitors demonstrate low nM inhibitory potency for pKal and varying specificity for related serine proteases. Compound 1 utilizes a surprising mode of interaction and upon binding results in a rearrangement of the binding pocket. Co-crystal structures of pKal describes why this class of small-molecule inhibitor is potent. Lack of conservation in surrounding residues explains the ∼10,000-fold specificity over structurally similar proteases, as shown by in vitro protease inhibition data. Structural information, combined with biochemical and enzymatic analyses, provides a novel scaffold for the design of targeted oral small molecule inhibitors of pKal for treatment of HAE and other diseases resulting from unregulated plasma kallikrein activity.

Plasma kallikrein mediates brain hemorrhage and edema caused by tissue plasminogen activator therapy in mice after stroke.

Thrombolytic therapy using tissue plasminogen activator (tPA) in acute stroke is associated with increased risks of cerebral hemorrhagic transformation and angioedema. Although plasma kallikrein (PKal) has been implicated in contributing to both hematoma expansion and thrombosis in stroke, its role in the complications associated with the therapeutic use of tPA in stroke is not yet available. We investigated the effects of tPA on plasma prekallikrein (PPK) activation and the role of PKal on cerebral outcomes in a murine thrombotic stroke model treated with tPA. We show that tPA increases PKal activity in vitro in both murine and human plasma, via a factor XII (FXII)-dependent mechanism. Intravenous administration of tPA increased circulating PKal activity in mice. In mice with thrombotic occlusion of the middle cerebral artery, tPA administration increased brain hemorrhage transformation, infarct volume, and edema. These adverse effects of tPA were ameliorated in PPK (Klkb1)-deficient and FXII-deficient mice and in wild-type (WT) mice pretreated with a PKal inhibitor prior to tPA. tPA-induced brain hemisphere reperfusion after photothrombolic middle cerebral artery occlusion was increased in Klkb1-/- mice compared with WT mice. In addition, PKal inhibition reduced matrix metalloproteinase-9 activity in brain following stroke and tPA therapy. These data demonstrate that tPA activates PPK in plasma and PKal inhibition reduces cerebral complications associated with tPA-mediated thrombolysis in stroke.

Improving the understanding of plasma kallikrein contribution to arterial thrombus formation using two plant protease inhibitors.

The purpose of antithrombotic therapy is the prevention of thrombus formation and/or its extension with a minimum risk of bleeding. The inhibition of a variety of proteolytic processes, particularly those of the coagulation cascade, has been reported as a property of plant protease inhibitors. The role of trypsin inhibitors (TIs) from Delonix regia (Dr) and Acacia schweinfurthii (As), members of the Kunitz family of protease inhibitors, was investigated on blood coagulation, platelet aggregation, and thrombus formation. Different from Acacia schweinfurthii trypsin inhibitor (AsTI), Delonix regia trypsin inhibitor (DrTI) is a potent inhibitor of FXIa with a Kiapp of 1.3 × 10-9 M. In vitro, both inhibitors at 100 µg corresponding to the concentrations of 21 µM and 15.4 µM of DrTI and AsTI, respectively, increased approximately 2.0 times the activated partial thromboplastin time (aPTT) in human plasma compared to the control, likely due to the inhibition of human plasma kallikrein (huPK) or activated factor XI (FXIa), in the case of DrTI. Investigating in vivo models of arterial thrombus formation and bleeding time, DrTI and AsTI, 1.3 µM and 0.96 µM, respectively, prolonged approximately 50% the time for total carotid artery occlusion in mice compared to the control. In contrast to heparin, the bleeding time in mice treated with the two inhibitors did not differ from that of the control group. DrTI and AsTI inhibited 49.3% and 63.8%, respectively, ex vivo murine platelet aggregation induced by adenosine diphosphate (ADP), indicating that these protein inhibitors prevent arterial thrombus formation possibly by interfering with the plasma kallikrein (PK) proteolytic action on the intrinsic coagulation pathway and its ability to enhance the platelet aggregation activity on the intravascular compartment leading to the improvement of a thrombus.

Prevention of acute liver injury by suppressing plasma kallikrein-dependent activation of latent TGF-ß.

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor ß (TGF-ß) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-ß is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-ß activation. This study aimed to explore the potential roles of PLK-dependent TGF-ß activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-ß1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-ß activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-ß receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-ß activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-ß activation has potential as a therapeutic strategy for ALI.

A critical role for plasma kallikrein in the pathogenesis of autoantibody-induced arthritis.

The plasma kallikrein-kinin system (KKS) consists of serine proteases, prekallikrein (pKal) and factor XII (FXII), and a cofactor, high-MW kininogen (HK). Upon activation, activated pKal and FXII cleave HK to release bradykinin. Activation of this system has been noted in patients with rheumatoid arthritis, and its pathogenic role has been characterized in animal arthritic models. In this study, we generated 2 knockout mouse strains that lacked pKal and HK and determined the role of KKS in autoantibody-induced arthritis. In a K/BxN serum transfer-induced arthritis (STIA) model, mice that lacked HK, pKal, or bradykinin receptors displayed protective phenotypes in joint swelling, histologic changes in inflammation, and cytokine production; however, FXII-deficient mice developed normal arthritis. Inhibition of Kal ameliorated arthritis severity and incidence at early stage STIA and reduced the levels of major cytokines in joints. In addition to releasing bradykinin from HK, Kal directly activated monocytes to produce proinflammatory cytokines, up-regulated their C5aR and FcRIII expression, and released C5a. Immune complex increased pKal activity, which led to HK cleavage. The absence of HK is associated with a decrease in joint vasopermeability. Thus, we identify a critical role for Kal in autoantibody-induced arthritis with pleiotropic effects, which suggests that it is a new target for the inhibition of arthritis.-Yang, A., Zhou, J., Wang, B., Dai, J., Colman, R. W., Song, W., Wu, Y. A critical role for plasma kallikrein in the pathogenesis of autoantibody-induced arthritis.

Single-chain factor XII: a new form of activated factor XII.

PURPOSE OF REVIEW: Exposure of blood to foreign surfaces induces reciprocal conversion of the plasma proteins factor XII (fXII) and plasma prekallikrein (PPK) to the proteases α-fXIIa and α-kallikrein. This process, called contact activation, has a range of effects on host defence mechanisms, including promoting coagulation. The nature of the triggering mechanism for contact activation is debated. One hypothesis predicts that fXII has protease activity, either intrinsically or upon surface-binding, that initiates contact activation. We tested this by assessing the proteolytic activity of a recombinant fXII variant that cannot be converted to α-fXIIa. RECENT FINDINGS: The proteolytic activity of fXII-T (for 'triple' mutant), a variant with alanine substitutions for arginine at activation cleavage sites (Arg334, Arg344, and Arg353) was tested with known α-fXIIa substrates. FXII-T activates PPK in solution, and the reaction is enhanced by polyphosphate, an inducer of contact activation released from platelets. In the presence of polyphosphate, fXII-T converts fXII to α-fXIIa, and also converts the coagulation protein factor XI to its active form. SUMMARY: The findings support the hypothesis that contact activation is initiated through activity intrinsic to single-chain fXII, and indicate that preexisting α-fXIIa is not required for induction of contact activation.

Blocking of plasma kallikrein ameliorates stroke by reducing thromboinflammation.

OBJECTIVE: Recent evidence suggests that ischemic stroke is a thromboinflammatory disease. Plasma kallikrein (PK) cleaves high-molecular-weight kininogen to release bradykinin (BK) and is a key constituent of the proinflammatory contact-kinin system. In addition, PK can activate coagulation factor XII, the origin of the intrinsic coagulation cascade. Thus, PK triggers 2 important pathological pathways of stroke formation, thrombosis and inflammation. METHODS: We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke. RESULTS: PK-deficient mice of either sex challenged with transient middle cerebral artery occlusion developed significantly smaller brain infarctions and less severe neurological deficits compared with controls without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarctions as well as after permanent stroke. Reduced intracerebral thrombosis and improved cerebral blood flow could be identified as underlying mechanisms. Moreover, blood-brain barrier function was maintained in mice lacking PK, and the local inflammatory response was reduced. PK-deficient mice reconstituted with PK or BK again developed brain infarctions similar to wild-type mice. Important from a translational perspective, inhibition of PK in wild-type mice using a PK-specific antibody was likewise effective even when performed in a therapeutic setting up to 3 hours poststroke. INTERPRETATION: PK drives thrombus formation and inflammation via activation of the intrinsic coagulation cascade and the release of BK but appears to be dispensable for hemostasis. Hence, PK inhibition may offer a safe strategy to combat thromboembolic disorders including ischemic stroke.

Plasma kallikrein-bradykinin pathway promotes circulatory nitric oxide metabolite availability during hypoxia.

Nitric oxide (NO) is an indispensible signalling molecule under hypoxic environment for both ethnic high altitude natives as well as lowland residents at high altitude. Several studies have reported higher levels of NO and bioactive NO products for both high altitude natives as well as healthy high altitude sojourners. But the metabolic pathways regulating the formation of NO and associated metabolites during hypoxia still remain elusive. In the present study, we profiled plasma proteomes of Ladakhi natives (3520 m) and lowland residents (post 1, 4 and 7 days stay) at the same altitude. This has resulted in the identification of 208 hypoxia responsive proteins (p < 0.05) and kininogen-plasma kallikrein-bradykinin as a major pathway regulating eNOS activity during hypoxia. In corroboration, we have also observed significant higher levels of plasma biomarkers for NO production (l-citrulline, nitrite, nitrate) for Ladakhi natives as compared to both lowland individuals healthy high altitude sojourners indicating higher NO availability. Since hypoxia-induced free radicals reduce NO availability, we also measured plasma levels of 8-isoprostanes, protein carbonyls and protein oxidation products in both Ladakhi natives and high altitude sojourners. Interestingly Ladakhi natives had significant lower levels of oxidative stress in comparison to high altitude sojourners but higher than lowland controls. These results suggest that plasma kallikrein-bradykinin-eNOS pathway along with moderate oxidative stress contributes to high altitude adaptation of Ladakhi natives.

Diagnostic Accuracy of Serum Kallikrein-Related Peptidases for Ovarian Cancer: A Systematic Review and Meta-Analysis.

OBJECTIVE: At present, considerable efforts have been made to identify new cancer-specific markers for ovarian cancer (OC) diagnosis and the kallikrein-related peptidases (KLKs) family is one of the most studied candidates. This meta-analysis aims to evaluate the pooled diagnostic value of serum KLK measurement for diagnosing OC. METHODS: The Cochrane Library, PubMed, Excerpt Medica Database were searched for all relevant literature. The Quality Assessment for Studies of Diagnostic Accuracy tool was applied to assess the quality of enrolled studies. Statistical analysis was conducted by using Stata 13.0 software and Meta-Disc. RESULTS: A total of 15 studies from 13 articles were considered eligible for inclusion in the present analysis. The following pooled parameters were calculated by using the bivariate model: sensitivity of 0.582 (95% confidence interval [CI], 0.517-0.644), specificity of 0.909 (95% CI, 0.833-0.952), positive likelihood ratios of 6.367 (95% CI, 3.330-12.172), negative likelihood ratios of 0.460 (95% CI, 0.388-0.546), diagnostic odds ratio of 13.831 (95% CI, 6.460-29.614), respectively. CONCLUSIONS: Kallikrein-related peptidase seems to be a promising candidate biomarker in diagnosing OC, but the associated poor sensitivity of KLK individually may limit its value in clinical application. To resolve this problem, the combination of KLK and other markers may offer improved performance than a single marker.

Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels.

The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17 × 10(-7)), which was also observed in a COPD population (combined P=5.04 × 10(-12)). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases.

Interleukin-like epithelial-to-mesenchymal transition inducer activity is controlled by proteolytic processing and plasminogen-urokinase plasminogen activator receptor system-regulated secretion during breast cancer progression.

INTRODUCTION: Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive. METHODS: The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients. RESULTS: We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer. CONCLUSIONS: Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.

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

ABSTRACT: Plasma 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.

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.

Extension of the circulatory half-life of recombinant ecallantide via albumin fusion without loss of anti-kallikrein activity.

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (Ki) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. OBJECTIVES: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. METHODS: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. RESULTS: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. CONCLUSION: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

[Kallikrein-kinin system as a target for diabetic retinopathy treatment].

Multifactor etiology of diabetic retinopathy (DR) determines difficulty of understanding of pathogenesis and need of search of effective approaches to study key mechanisms of development of this microvascular complication of diabetes mellitus (DM). Significant achievements of the last years show the contribution of two proteolytic systems into pathogenesis of DR, that control vascular tone and permeability - kallikrein-kinin (KKS) and renin-angiotensin systems (RAS). Among new approaches to DR treatment one of the most appropriate is an influence on KKS by means of inhibiting kallikrein, that leads to reduction of retinal vascular permeability and allows to prevent the development of macula oedema and other consequences of vascular wall damage in DR.