In the absence of PKa-i, EA-triggered thrombin generation in FXI-deficient plasma was not influenced by inhibition of the TF-FVII pathway (Figure 2B). In contrast to the results observed in FXI-deficient plasma, PKa-i did not influence EA-induced thrombin generation in normal pooled plasma (Figure 1B graph 1). 1-800-AHA-USA-1 Factor V: Labile factor (Leads), Proaccelerin 6. The inhibitory potential of the PKa-i towards murine PKa was validated in a pilot experiment using plasma from FXI-deficient animals (F11−/−). Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation. The inhibitory potencies of the PKa-i on PKa were additionally determined in pooled human plasma collected from 10 healthy donors. polyP indicates polyphosphate. Ab indicates antibody; aPTT, activated partial thromboplastin time; def., deficient; and depl., depleted. Patients with severe factor XI deficiency have a reduced incidence of deep-vein thrombosis. PK drives multiple proteolytic reaction cascades in the cardiovascular system such as the intrinsic pathway of coagulation, the kallikrein-kinin system, the fibrinolytic system, the renin-angiotensin … Cate received reimbursement for advisory boards from Pfizer, Boehringer, Leo farma, and Daiichi. Data were pooled for analysis to obtain a higher total number of animals in the groups, as the data showed no differences between male and female animals. These findings may, in part, explain the different phenotypes associated with FIX and FXI deficiencies. C, Plasma with different FXI concentrations was prepared by mixing normal plasma with FXI-deficient plasma. Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule. It is enzymatically inactive and functions as a cofactor for the activation of kallikrein … Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma. Dual role of collagen in factor XII-dependent thrombus formation. Nolly H, Miatello R, Damiani MT, Abate CD. Because the in vitro experiments show that the effect of PKa inhibition on fibrin formation was more pronounced in the absence of FXI, F11−/− mice instead of wild-type mice were used in the in vivo experiments. Cate was a Fellow of the Gutenberg Research Foundation. 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. A value of P<0.05 was considered statistically significant. Addition of a specific active-site inhibitor of PKa caused a dose-dependent reduction of thrombin generation, a prolongation of aPTT and a delay of FIX-AT and FX-AT complex formation in FXI-deficient plasma in both, artificial and physiological settings. coagulation, complement and fibrinolytic systems in the plasma of six patients with HAE caused by the Thr309Lys mutation and healthy probands. def. Pefabloc FG (4.5 mg/mL; Pentapharm, Switzerland) was added to each sample to prevent clot formation. FXIa (factor XIa) induces clot formation, and human congenital FXI deficiency protects against venous thromboembolism and stroke. Kallikrein (PKa), generated by acti vation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. Similar results were observed when contact activation was induced with long-chain polyphosphate. Pharmacological enhancement of the kallikrein-kinin system promotes anti-fibrotic responses in human mesangial cells. Patients with unstable angina pectoris had a significantly (P<.05) increased plasma kallikrein activity up to 46.0±5.3 U/L. To investigate the potential activation of FIX by PKa we conducted both in vitro and in vivo experiments. Recent evidence suggests that the coagulation and kallikrein-kinin cascade might participate in this process. It also participates in the contact phase of blood coagulation. 10 µL per sample was taken at 5, 10, 20, 30, 40, 50, and 60 minutes after initiation of contact activation and mixed with 10 µL 3.2% (w/v) sodium citrate to stop coagulation. After a 3-minute incubation at 37°C, a kallikrein-specific substrate (50 µmol/L, I-1295; Bachem) was added, and fluorescence was measured for 40 minutes at 37°C (excitation: 360 nm; emission: 460 nm). Thrombin generation in FXI-deficient plasma was completely inhibited by PKa-i (45 µmol/L BAY-992). In line with this, Osterud et al16 calculated that FXI is ≈20 000× more active than PKa in activating FIX under the conditions they used for their experiments. PKa (plasma kallikrein) is involved in FIXa (activated factor IX)-AT (antithrombin) complex formation in the presence of calcium ions in FXI-deficient plasma. Upon initiation of thrombin generation with EA, FIXa-AT complex levels instantly reached the upper detection limit of 1.6 nmol/L. Formation of the clot. Giovanella J, Wollinger LM, Capra L, Dresch F, Genro JP, Contini V. Mol Cell Biochem. The data that support the findings of this study are available from the corresponding author on reasonable request. Kallikrein is a serine protease which consists of a heavy chain (Mr 52kD) and light chains (Mr either 36 or 33 kD) linked by disulfide bridges. Because FIX seems to be activated via the Josso-loop in the extrinsic pathway, by FXI in the intrinsic pathway, and by PKa, the enzyme FIX might be considered as a key regulatory coagulation protease. National Library of Medicine The relative PKa inhibition by the inhibitors was calculated, and IC50 values were determined as described above. XIIa, kallikrein… Addition of PKa-i dose-dependently delayed FIXa-AT complex formation for 20 min at an inhibitor concentration of 15 µmol/L, with complete inhibition at a concentration of 150 µmol/L PKa-i (Figure 3A). 2020;40:103–111. In both normal and plasma prekallikrein-depleted plasma, addition of a PKa-i prolonged the aPTT by a factor 1.2 (Figure 1A). The first experimental evidence for this was provided by Osterud et al16 in 1978, demonstrating that PKa can activate FIX under the conditions of an artificial, purified system. These results indicate that PKa may activate FIX in the absence of FXI and that calcium is required for the formation of FIXa-AT complexes. These data suggest that, in the absence of FXI, PKa may activate FX. Local Info Effects of plasma kallikrein deficiency on haemostasis and thrombosis in mice: murine ortholog of the fletcher trait. Plasma kallikrein functions in the vascular space and is responsible for the formation of bradykinin. Factor IV: Calcium (Cancer) 5. The kallikrein-kinin system and oxidative stress. B, Thrombin generation was not influenced by active-site–inhibited FVIIa (ASIS; 45 nmol/L) alone but was reduced in combination with PKa-i (0.45–45 µmol/L BAY-992). Central regulator of hemostasis. Our in vivo data indicate that the FXI bypass pathway of FIX activation by PKa exists in mice. Coagulation factor XII cleaves prekallikrein, and the generated Plasma kallikrein catalyses Bradykinin formation and activates Coagulation factor XI . Samples were analyzed for activity and concentrations of components of the kallikrein… 2020 Sep 11;11:883. doi: 10.3389/fgene.2020.00883. Effect of combining low levels of tissue factor with complete deficiencies for factor IX or factor XI on survival and placental development in mice. The plasma kallikrein–kinin system consists of the proteins factor XII (FXII), … ), Laboratory for Clinical Thrombosis and Haemostasis, Departments of Biochemistry and Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands (M.V., R.v.O., H.t.C., H.M.H.S. E, Thrombin generation in FXI-deficient plasma was initiated by 2.5 mmol/L polyphosphate, 4 µmol/L phospholipids and calcium chloride in the presence or absence of PKa-i (0.45–4.5 µmol/L BAY-992). Summary. Factor VIII: Ant… Correspondence to: Stefan Heitmeier, PhD, Research and Development, Bayer AG, 42096 Wuppertal, Germany. Plasma kallikrein-like activity was … FIXa-AT complex formation was increased by EA compared with control (plasma w/o EA), and this increase was prevented by PKa-i (plasma kallikrein inhibitor; 45 µmol/L BAY-077). In the past, it had been shown that enzymes of the coagulation cascade may act on several different zymogens or cofactors. In-house FIXa-AT or FXa-AT inhibitor complex ELISA was used to determine the amount of FIXa or FXa, as described previously.19 In brief, sheep anti-human FIX or sheep anti-human FX (2 µg/mL, Affinity Biologicals, Canada) was used as capture antibody, whereas biotin-labeled sheep anti-human AT antibodies (1 µg/mL, Affinity Biologicals) were applied as detection antibody. For these experiments, contact activation in congenital FXI-deficient plasma was initiated by 2.5 mmol/L polyphosphate, 4 µmol/L phospholipids and calcium chloride in the presence or absence of PKa-i (0.45–4.5 µmol/L). Factor XI in haemostasis and thrombosis: past, present and future. To extend the in vitro data showing a role for PKa in FIX activation in human plasma, we investigated if similar results could be observed in mice. FIXa-AT complex formation was slower than in EA-initiated contact activation, but the upper detection limit was also reached after 30 minutes. 2005 Jan;3(1):33-44. doi: 10.2174/1568016052773351. Without the addition of calcium chloride, no FIXa-AT complex formation could be observed (Figure 3B). It serves as a critical cofactor for the prothrombinase activity of factor Xa that results in the activation of prothrombin to … Cate receive support from the Netherlands Heart Foundation: CVON2014-09, RACE V (Reappraisal of Atrial Fibrillation: Interaction between HyperCoagulability, Electrical Remodeling, and Vascular Destabilisation in the Progression of Atrial Fibrillation). For example, thrombin not only converts fibrinogen to fibrin but also activates FXI, FV, FVIII, and protein C (Figure 5; reviewed by Lane et al).31 The TF-FVIIa complex of the extrinsic pathway activates not only FX but also FIX in the so-called Josso-loop (Figure 5).32,33 FX has always been considered as the predominant enzyme of the common pathway merging the 2 coagulation pathways. Human blood coagulation factor IX. Cell Physiol Biochem. PKa (plasma kallikrein) is involved in the contact activation pathway initiated by artificial surfaces or long-chain polyphosphate in the absence of FXI (factor XI). Comparison of groups of the in vivo experiment was performed using 1-way ANOVA and the Holm-Sidak test for multiple comparisons in Prism 8 (Graphpad, CA). In the absence of calcium, no FIXa-AT complex levels were measured, suggesting that calcium is either essential for the activation of FIX by PKa or for the measurement of FIXa-AT complexes. Coagulation Factor XII is responsible for the conversion of prekallikrein into active plasma kallikrein [26]. Accessibility D, FXa-AT complex formation in FXI-deficient plasma was initiated with 100 µg/mL ellagic acid, 4 µmol/L phospholipids, and calcium chloride in the presence or absence of the PKa-i BAY-992. Samples were taken at defined time points. FXI consists of 2 identical subunits, each comprising 4 tandem repeats of each 90 to 91 amino acids, named apple domains (A1–A4), and a trypsin-like catalytic domain.15,26 The amino acid sequence of plasma prekallikrein is 58% identical to that of FXI, and the zymogen contains the characteristic 4 apple domains.8,27 Besides the sequence homology, both proteins mostly circulate in a noncovalent complex with HK,28,29 facilitating the activation of both zymogens by FXIIa.30. The contact system, also named as plasma kallikrein-kinin system, consists of three serine proteinases: coagulation factors XII (FXII) and XI (FXI), and plasma prekallikrein (PK), and the … All animal experiments were conducted in accordance to the Guidelines of the European (Guideline 2010/63/EU) and National Legislation (dt. *These authors contributed equally to this article. Furthermore, an antibody directed against the active site of FXIa (Bayer AG, described previously)18 was used at a plasma concentration of 4.5 µmol/L to verify FXI deficiency of the used plasma. Moreover, polyphosphate-induced contact activation in FXI-deficient plasma was dose-dependently inhibited by PKa-i (Figure 1E through 1G). FXIIa was unable to induce clotting in FXI-deficient plasma in the presence of PKa-i, excluding the possibility of FIX activation by FXIIa (Figure 2C). Figure 2. F, The lag time of polyphosphate-induced thrombin generation in FXI-deficient plasma was prolonged and (G) peak height was decreased in the presence of PKa-i. Prest, Diagnostica Stago). Data were not tested for normality and equal variance. Mice were randomly divided into different groups (control, EA and EA+PKa-i group or control, polyphosphate and polyphosphate+PKa-i group) and anesthetized by intraperitoneal injection of 10 µL/g mixture of 11.1 mg/mL Ketamin (Ketaset, Zoetis, NJ) and 0.11% (w/v) Xylazine (Rompun, Bayer Vital, Germany). By continuing to browse this site you are agreeing to our use of cookies. However, the question about the cause of the different phenotypes associated with FXI deficiency and FIX or FVIII deficiency still remains. This leads to activation of FXI to FXIa, which in turn converts FIX to FIXa, eventually leading to thrombin generation and fibrin formation. Addition of PKa-i reduced EA-triggered thrombin generation in the absence of FXI, with complete inhibition at a concentration of 150 µmol/L PKa-i (Figure 1B through 1D), which was also observed in kaolin-triggered plasma (Figure VB in the online-only Data Supplement). NX_P12259 - F5 - Coagulation factor V - Function. Please enable it to take advantage of the complete set of features! The control group received 0.9% (w/v) saline and Pefabloc FG (9 mg/kg) only. Tierschutzgesetz v. 04.07.2013) for the use and protection of animals for scientific purposes and approved by the institutional animal care office of Bayer AG and the competent regional authority (LANUV NRW, permit no. 84-02.05.40.17.12). The main function of kallikrein in … In plasma with 1% FXI level, however, PKa-i prolonged the lag time but did not reduce peak thrombin. Blood samples were taken from participants during the symptom-free interval between attacks. B, EA (30 mg/kg) or (C) long-chain polyphosphate (1 µL/g of 107 mmol/L polyphosphate) and Pefabloc FG (9 mg/kg) with or without the PKa-i BAY-077 (20 mg/kg in EA group; 10 mg/kg in polyphosphate group) were injected intravenously into F11−/− mice. Overestimation of N-glycoPEGylated factor IX activity in a one-stage factor IX clotting assay owing to silica-mediated premature conversion to activated factor IX. This article was sent to Karlheinz Peter, Consulting Editor, for review by expert referees, editorial decision, and final disposition. High-molecular-weight kininogen (HMWK) is also called as the Williams-Fitzgerald-Flaujeac factor. 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. Using a buffer system, Osterud et al16 showed that PKa activates FIX. 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. Compared with FIX or FVIII deficiency, FXI-deficient subjects exhibit, in general, a mild bleeding phenotype with very rare spontaneous bleedings.10 The likelihood of bleeding is increased after trauma or surgery, especially if tissues with high fibrinolytic activity are affected.10,12 However, the bleeding tendency of FXI-deficient subjects is, unlike FIX or FVIII deficiency, not correlated with the FXI level.11 It has been suggested that other hemostatic abnormalities including low von Willebrand factor level contribute to the bleeding risk in FXI-deficient subjects.13 We hypothesize that reductions in plasma prekallikrein levels or activity in FXI-deficient subjects might lead to a higher bleeding risk due to diminished activation of FIX. A, Contact activation in FXI-deficient plasma was induced with 100 µg/mL ellagic acid, 4 µmol/L phospholipids, and calcium chloride in the presence or absence of the PKa-i (PKa inhibitor; BAY-992). Factor XII activation promotes platelet consumption in the presence of bacterial-type long-chain polyphosphate. We recently demonstrated that FIX can be activated by TF (tissue factor)-FVIIa (Josso-Loop) in mice (Mackman et al14). eCollection 2020. Coagulation and Kallikrein-Kinin System . 1997 Jun;36(2-3):185-91. doi: 10.1016/s0162-3109(97)00020-9. Human blood coagulation factor XI. Without addition of EA or polyphosphate, coagulation was not initiated, and addition of an FXIa antibody had no effect on thrombin generation. Curr Med Chem Cardiovasc Hematol Agents. Data are presented as the means±SD (n=3). In support of this, plasma prekallikrein–deficient mice showed reduced thrombus formation in both arterial and venous thrombosis models.34–36. 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. Front Genet. Ellagic acid (EA) increases FIXa (activated factor FIX)-AT (antithrombin) complex formation in plasma from F11−/− mice, and EA and long-chain polyphosphate induce FIXa-AT complex formation in F11−/− mice. [1] It consists of blood … Contact activation was induced by addition of a kaolin-cephalin suspension (C.K. These studies show that FXI plays a role in thrombosis and might be a valuable target to prevent thrombosis. The regulation of this system by serpins and the wide distribution of the different constituents add to the complexity of this system, as well as its multiple relationships with other important metabolic pathways such as the renin-angiotensin, coagulation, or complement pathways. The Kallikrein-Kinin System in Coagulation The kallikrein-kinin system comprises a complex of proteins that when activated leads to the release of vasoactive kinins. C, Contact activation in FXI-deficient plasma was induced with 2.5 mmol/L long-chain polyphosphate, 4 µmol/L phospholipids, and calcium chloride. High levels of coagulation factor XI as a risk factor for venous thrombosis. H.M.H. Crystal structure of the factor XI zymogen reveals a pathway for transactivation. We demonstrated that activation of FXII leads to thrombin generation via FIX activation by PKa in the absence of FXI. The IC50 values towards human PKa in human plasma were 1.3 µmol/L for BAY-992 and 0.2 µmol/L for BAY-077 (Figure II in the online-only Data Supplement). However, FXIIa-activation of FXI to FXIa, which in turn activates FIX, is most likely the more dominant pathway, as specific inhibition of PKa did not inhibit FIXa generation in normal plasma compared with the attenuation observed in FXI-deficient plasma. Bethesda, MD 20894, Copyright COVID-19 is an emerging, rapidly evolving situation. Cate and H.M.H. Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III. In conclusion, we demonstrate that in the absence of FXI, activation of FXII by EA or long-chain polyphosphates leads to thrombin generation via FIX activation by PKa. Thrombin generation was initiated as described above in the presence or absence of PKa-i (45 µmol/L). Figure 3. The anti-factor XIa antibody BAY 1213790 is a novel anticoagulant that shows strong antithrombotic efficacy without an increased risk of bleeding in rabbit models. 2006;18(6):327-36. doi: 10.1159/000097610. First, the contribution of PKa to the contact activation system was estimated using the aPTT assay in 3 different plasma types: normal human, plasma prekallikrein-depleted, and FXI-deficient plasma, respectively. A, PKa inhibition (PKa-i [PKa inhibitor]: 0.185–45 µM BAY-992) dose-dependently prolonged kaolin-triggered clotting time in human congenital FXI-deficient plasma but not in normal pooled or plasma prekallikrein-depleted plasma. Data are presented as the means±SD (n=3). These results indicate that FIX is not activated by FXIIa and that sufficient FXII is formed by EA in the presence of PKa-i. In addition, FXIIa does not activate FIX. Activation of the intrinsic pathway of coagulation, in part, involves the contribution of the kallikrein … Diet-gene interaction: effects of polymorphisms in the ACE, AGT and BDKRB2 genes and the consumption of sodium, potassium, calcium, and magnesium on blood pressure of normotensive adult individuals. The kallikrein-kinin system is an endogenous metabolic cascade, triggering of which results in the release of vasoactive kinins (bradykinin-related peptides). Data are presented as the means±SD. Because formation of FXa-AT complexes could be observed in EA-induced contact activation in FXI-deficient plasma, we investigated if FIXa might be formed by using an in-house ELISA. Clipboard, Search History, and several other advanced features are temporarily unavailable. Careers. Immunopharmacology. Thrombin generation measurement by means of the Calibrated Automated Thrombogram (Stago, France) was performed as described previously.17 In brief, the contact activation system in congenital FXI-deficient plasma (George King Bio-Medical, Inc) was induced by addition of 100 µg/mL EA (Sigma-Aldrich, MO), 4 µmol/L phospholipids at 20:20:60 mol% PS:PE:PC (phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine) and calcium chloride. In addition, the importance of this complex is the anticoagulant function of antibodies to this receptor (eg, abciximab: see below). From the Bayer AG, Cardiovascular Research, Wuppertal, Germany (M.V., V.L., S.H. Unauthorized PKa-i were serially diluted and mixed with the human purified enzyme (thrombin, FXa, FXIIa, FXIa, plasmin, tissue-type plasminogen activator, and trypsin; Enzyme Research Laboratories, IN), and its specific substrate (Bachem, Switzerland). Upon initiation of thrombin generation in FXI-deficient plasma, FXa-AT complex levels were immediately above the upper detection limit of 1.6 nmol/L. Our goal was to evaluate whether PKa contributes to the FXIa-independent activation of FIX in human plasma and in mice. C, The lag time of ellagic acid–induced thrombin generation in FXI-deficient plasma was prolonged, and (D) peak height was decreased by inhibition of PKa.