ecallantide and Disease-Models--Animal

Cardiovascular disease, including stroke, myocardial infarction, and venous thromboembolism, is one of the leading causes of morbidity and mortality worldwide. Excessive coagulation may cause vascular occlusion in arteries and veins eventually leading to thrombotic diseases. Studies in recent years suggest that coagulation factors are involved in these pathological mechanisms. Factors XIa (FXIa), XIIa (FXIIa), and plasma kallikrein (PKa) of the contact system of coagulation appear to contribute to thrombosis while playing a limited role in hemostasis. Contact activation is initiated upon autoactivation of FXII on negatively charged surfaces. FXIIa activates plasma prekallikrein (PK) to PKa, which in turn activates FXII and initiates the kallikrein-kinin pathway. FXI is also activated by FXIIa, leading to activation of FIX and finally to thrombin formation, which in turn activates FXI in an amplification loop. Animal studies have shown that arterial and venous thrombosis can be reduced by the inhibition of FXI(a) or PKa. Furthermore, data from human studies suggest that these enzymes may be valuable targets to reduce thrombosis risk. In this review, we discuss the structure and function of FXI(a) and PK(a), their involvement in the development of venous and arterial thrombosis in animal models and human studies, and current therapeutic strategies.

Topics: Animals; Arterial Occlusive Diseases; Blood Coagulation; Blood Coagulation Disorders; Blood Coagulation Factors; Disease Models, Animal; Enzyme Activation; Factor Xa Inhibitors; Factor XI Deficiency; Factor XIa; Humans; Mice; Mice, Knockout; Plasma Kallikrein; Prekallikrein; Protein Processing, Post-Translational; Species Specificity; Thrombophilia; Thrombosis; Venous Thrombosis

To ascertain the participation of the plasma kallikrein-kinin system (KKS) in arthritis and inflammatory bowel disease, we used two rat models resembling rheumatoid arthritis and Crohn's disease. Proteoglycan-polysaccharide from group A streptococcus (PG-APS) produced chronic destructive inflammation and systemic response in the genetically susceptible Lewis rat, in the joints when injected intraperitoneally and in the bowel when injected into the gut wall. In both models, the KKS is activated, as evidenced by decreased prekallikrein, factor XI and high molecular weight kininogen. A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroarginine, reverses the plasma changes as well as the clinical gross and microscopic pathology of both the experimental arthritis and the inflammatory bowel disease in the genetically susceptible rats. We have also shown that the tissue kallikrein system is involved in the intestinal inflammatory changes. Intestinal tissue kalikrein (ITK) is localized in goblet cells in both normal and inflamed tissue. In chronic granulomatous inflammation, ITK is localized in macrophages. ITK decreases in chronic inflammation, probably due to secretion, since the mRNA is unchanged. Kallikrein binding protein, the ITK inhibitor, decreases due to enzyme-inhibitor complexes. Both plasma and tissue kallikrein are appealing targets for drug therapy of rheumatoid arthritis and Crohn's disease.

Topics: Animals; Arthritis, Rheumatoid; Disease Models, Animal; Humans; Inflammatory Bowel Diseases; Intestines; Plasma Kallikrein; Polysaccharides, Bacterial; Proteoglycans; Rats; Rats, Inbred Lew; Tissue Kallikreins

Hematoma expansion (HE) aggravates brain injury after intracerebral hemorrhage (ICH) and hypertension is a key contributor to HE. Plasma kallikrein (PK) is involved in hemorrhagic transformation in ischemic stroke mice. This study was conducted to explore the role of PK in HE in hypertensive ICH.. Hypertension was achieved by continuous infusion of angiotensin II (Ang II) with an osmotic pump in C57BL/6 mice. ICH was achieved by stereotactic intrastriatal injection of blood. PK-specific antibody and platelet glycoprotein VI (GPVI) agonists were administered to intervene in hematoma expansion. The hematoma volume was indicated by the erythrocyte components hemoglobin and carbonic anhydrase-1 in the ipsilateral brain hemisphere.. Ang II-induced hypertensive mice showed enhanced hematoma expansion and worsened neurologic deficits after ICH modeling. Moreover, intrastriatal injection of blood from Ang II-treated mice into normal mice increased the area of secondary hemorrhage more than blood from untreated mice. Mechanistically, elevated PK was found in Ang II-infused mice whereas, inhibition of PK and administration of the GPVI agonist convulxin decreased hematoma expansion and improved neurologic deficits after ICH.. These findings suggest that PK inhibition and GPVI agonist treatment might serve as potential methods to intervene in HE after ICH.

Topics: Angiotensin II; Animals; Blood Pressure; Cerebral Hemorrhage; Crotalid Venoms; Disease Models, Animal; Hypertension; Lectins, C-Type; Male; Mice; Plasma Kallikrein; Treatment Outcome

Hereditary Angioedema (HAE) is a rare genetic disease generally caused by deficiency or mutations in the C1-inhibitor gene, SERPING1, a member of the Serpin family. HAE results in acute attacks of edema, vasodilation, GI pain and hypotension. C1INH is a key inhibitor of enzymes controlling complement activation, fibrinolysis and the contact system. In HAE patients, contact system activation leads to uncontrolled production of bradykinin, the vasodilator responsible for the characteristic symptoms of HAE. In this study, we present the first physiological in vivo model to mimic acute HAE attacks. We evaluate hypotension, one of the many hallmark symptoms of acute HAE attacks using Serping1 deficient mice (serping1-/-) and implanted telemetry. Attacks were induced by IV injection of a silica nanoparticle (SiNP) suspension. Blood pressure was measured in real time, in conscious and untethered mice using implanted telemetry. SiNP injection induced a rapid, reversible decrease in blood pressure, in the presence of angiotensin converting enzyme (ACE) inhibition. We also demonstrate that an HAE therapeutic, ecallantide, can prevent HAE attacks in this model. The in vivo murine model described here can facilitate the understanding of acute HAE attacks, support drug development and ultimately contribute to improved patient care.

Topics: Angioedemas, Hereditary; Animals; Bradykinin; Complement Activation; Complement C1 Inhibitor Protein; Disease Models, Animal; Edema; Female; Fibrinolysis; Hypotension; Male; Mice; Mice, Inbred C57BL; Peptides; Serpins

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. 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.

Topics: Animals; Blood Coagulation; Disease Models, Animal; Factor IX; Factor XI; Factor XI Deficiency; Female; Male; Mice; Mice, Inbred C57BL; Plasma Kallikrein; Thrombin; Thrombosis

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 K

Topics: Animals; Disease Models, Animal; Humans; Mice; Plants; Plasma Kallikrein; Protease Inhibitors; Thrombosis

Recent work has demonstrated that activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases causes sodium retention in nephrotic syndrome. The aim of this study was to elucidate a potential role of plasma kallikrein (PKLK) as a candidate serine protease in this context.. We analysed PKLK in the urine of patients with chronic kidney disease (CKD, n = 171) and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in PKLK-deficient mice (klkb1. In patients with CKD, we found that PKLK is excreted in the urine up to a concentration of 2 μg mL. Plasma kallikrein is detected in the urine of proteinuric patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site. However, PKLK is not essential for volume retention in nephrotic mice.

Topics: Adult; Aged; Animals; Body Composition; Case-Control Studies; Disease Models, Animal; Doxorubicin; Epithelial Sodium Channels; Female; Humans; Kidney; Male; Membrane Potentials; Mice, Knockout; Middle Aged; Natriuresis; Nephrotic Syndrome; Organism Hydration Status; Plasma Kallikrein; Prospective Studies; Renal Elimination; Renal Insufficiency, Chronic; Water-Electrolyte Balance; Xenopus laevis

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β 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-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β 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-β1 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-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β 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-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI.

Topics: Acetaminophen; Acute Lung Injury; Animals; Antibodies, Monoclonal; Benzamides; Biomarkers; Chemical and Drug Induced Liver Injury; Dioxoles; Disease Models, Animal; fas Receptor; Latent TGF-beta Binding Proteins; Macrophages; Male; Mice, Inbred C57BL; Plasma Kallikrein; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

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

Topics: Administration, Intravenous; Angioedema; Animals; Cerebral Hemorrhage; Disease Models, Animal; Factor XII; Fibrinolytic Agents; Gene Expression; Humans; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Plasma Kallikrein; Stroke; Thrombolytic Therapy; Thrombosis; Tissue Plasminogen Activator

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an

Topics: Animals; Caesalpinia; Cathepsin G; Disease Models, Animal; Kininogens; Models, Biological; Neutrophils; Phytochemicals; Plasma Kallikrein; Pneumonia; Protease Inhibitors; Rats; Seeds

Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulation factor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulation factors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

Topics: Angioedemas, Hereditary; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Coagulation; Capillary Permeability; Complement C1 Inhibitor Protein; Disease Models, Animal; Factor VII; Factor XI; Factor XII; Humans; Injections, Subcutaneous; Kinins; Male; Mice; Mice, Inbred BALB C; Oligonucleotides, Antisense; Plasma Kallikrein; Prekallikrein

The plasma kallikrein-mediated proteolysis regulates both thrombosis and inflammation. Previous study has shown that PF-04886847 is a potent and competitive inhibitor of kallikrein, suggesting that it might be useful for the treatment of kallikrein-kinin mediated inflammatory and thrombotic disorders. In the rat model of lipopolysaccharide (LPS) -induced sepsis used in this study, pretreatment of rats with PF-04886847 (1 mg/kg) prior to LPS (10 mg/kg) prevented endotoxin-induced increase in granulocyte count in the systemic circulation. PF-04886847 significantly reduced the elevated plasma 6-keto PGF1α levels in LPS treated rats, suggesting that PF-04886847 could be useful in preventing hypotensive shock during sepsis. PF-04886847 did not inhibit LPS-induced increase in plasma TNF-α level. Pretreatment of rats with PF-04886847 prior to LPS did not attenuate endotoxin-induced decrease in platelet count and plasma fibrinogen levels as well as increase in plasma D-dimer levels. PF-04886847 did not protect the animals against LPS-mediated acute hepatic and renal injury and disseminated intravascular coagulation (DIC). Since prekallikrein (the zymogen form of plasma kallikrein) deficient patients have prolonged activated partial thromboplastin time (aPTT) without having any bleeding disorder, the anti-thrombotic property and mechanism of action of PF-04886847 was assessed. In a rabbit balloon injury model designed to mimic clinical conditions of acute thrombotic events, PF-04886847 reduced thrombus mass dose-dependently. PF-04886847 (1 mg/kg) prolonged both aPTT and prothrombin time (PT) in a dose-dependent manner. Although the findings of this study indicate that PF-04886847 possesses limited anti-thrombotic and anti-inflammatory effects, PF-04886847 may have therapeutic potential in other kallikrein-kinin mediated diseases.

Topics: Aminobenzoates; Aminopyridines; Animals; Blood Coagulation; Disease Models, Animal; Disseminated Intravascular Coagulation; Dose-Response Relationship, Drug; Inflammation Mediators; Lipopolysaccharides; Male; Plasma Kallikrein; Rabbits; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Sepsis; Thrombosis

Plasma kallikrein is a multifunctional serine protease involved in contact activation of coagulation. Deficiency in humans is characterised by prolonged activated partial thromboplastin time (aPTT); however, the balance between thrombosis and haemostasis is not fully understood. A study of plasma kallikrein-deficient mice revealed increased aPTT, without prolonged bleeding time. Prekallikrein antisense oligonucleotide (ASO) treatment in mice suggested potential for a positive therapeutic index. The current goal was to further define the role of plasma kallikrein in coagulation. Blood pressure and heart rate were normal in plasma kallikrein-deficient mice, and mice were completely protected from occlusion (100 ± 1.3% control flow) in 3.5% FeCl3 -induced arterial thrombosis versus heterozygotes (20 ± 11.4%) and wild-type littermates (8 ± 0%). Vessels occluded in 8/8 wild-type, 7/8 heterozygotes, and 0/8 knockouts. Anti-thrombotic protection was less pronounced in 5% FeCl3-induced arterial injury. Integrated blood flow was 8 ± 0% control in wild-type and heterozygotes, and significantly (p<0.01) improved to 43 ± 14.2% in knockouts. The number of vessels occluded was similar in all genotypes. Thrombus weight was significantly reduced in knockouts (-47%) and heterozygotes (-23%) versus wild-type in oxidative venous thrombosis. Average tail bleeding time increased modestly in knockout mice compared to wild-type. Average renal bleeding times were similar in all genotypes. These studies confirm and extend studies with prekallikrein ASO, and demonstrate that plasma kallikrein deletion prevents occlusive thrombus formation in mice with a minimal role in provoked bleeding. Additional support for the significance of the intrinsic pathway in the coagulation cascade is provided, as well as for a potential new anti-thrombotic approach.

Topics: Animals; Bleeding Time; Chlorides; Disease Models, Animal; Ferric Compounds; Hemorrhage; Hemostasis; Heterozygote; Mice; Mice, Knockout; Oligonucleotides, Antisense; Partial Thromboplastin Time; Phenotype; Plasma Kallikrein; Prekallikrein; Thrombosis; Time Factors; Venous Thrombosis