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.

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Plasma Kallikrein Contributes to Ellagic Acid-Induced Coagulation in the Absence of FXI By Activating Factor IX

Blood ◽

10.1182/blood-2019-131738 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1106-1106

Author(s):

Mayken Visser ◽

Rene van Oerle ◽

Hugo ten Cate ◽

Volker Laux ◽

Stefan Heitmeier ◽

...

Keyword(s):

Complex Formation ◽

Ellagic Acid ◽

Plasma Levels ◽

Thrombin Generation ◽

Critical Role ◽

Factor Ix ◽

Coagulation Cascade ◽

Plasma Kallikrein ◽

Contact Activation ◽

The Impact

Background - Factor XI (FXI) plays a critical role in thrombosis, as its inhibition reduces clot formation in animal models, and human congenital FXI deficiency appears to protect against venous thromboembolism and stroke. In contrast, in both settings, the impact on hemostasis is rather low, especially compared to FIX deficiency. Little is known about the cause of this difference. As thrombin generation can be observed in FXI-deficient plasma, we speculated that intrinsic activation of FIX is not solely dependent on FXI(a). Aim - Evaluate whether plasma kallikrein contributes to FIX activation and subsequent thrombin generation in FXI deficiency. Methods - Kaolin-initiated coagulation and fibrin formation was assessed in different plasma types in the presence or absence of an active site inhibitor of human plasma kallikrein (PKa) using the activated partial thromboplastin time (aPTT). Thrombin generation was measured in ellagic acid-induced contact activation in human FXI-deficient plasma in the presence or absence of the PKa inhibitor. Levels of FIXa-antithrombin (FIXa-AT) or FXa-AT were determined at defined time points using in-house developed ELISAs. The role of thrombin and its substrate FVIII in ellagic acid-induced contact activation in the absence of FXI was investigated using dabigatran or a human monoclonal FVIII antibody and assessing the FIXa-AT and FXa-AT plasma levels. In in vivo experiments, F11-/- mice were treated with ellagic acid in the presence or absence of the PKa inhibitor and FXIa-AT levels were assessed 30 min after administration. Results - Plasma kallikrein inhibition dose-dependently prolonged kaolin-triggered clotting time in human FXI-deficient plasma but not in normal pooled or plasma prekallikrein depleted plasma, indicating a role for PKa in coagulation in the absence of FXI. In FXI-deficient plasma, ellagic acid activated the coagulation system, as could be demonstrated by measurement of thrombin generation, and FIXa-AT and FXa-AT levels, which immediately reached the upper detection limit of 1.6 nM suggesting a FXI bypass route towards FIX activation. Addition of a PKa inhibitor caused a delayed and diminished FXa-AT and FIXa-AT complex formation and led to a reduction in thrombin peak height (EC50: 3 µM). This indicates that PKa might contribute to FXI-independent coagulation by activating FIX. In the presence of dabigatran FIXa-AT complex formation in FXI-deficient plasma was completely inhibited, which could be reversed by addition of purified thrombin. In the presence of purified thrombin, a FVIII antibody inhibited thrombin-dependent FIXa-AT complex formation suggesting that the activation of FVIII by thrombin is essential for FIX activation by PKa or to support the activity of FIXa. In line with this, thrombin generation in FXI-defcient plasma was inhibited by the FVIII antibody. In ellagic acid-treated F11-/- mice, FIXa-AT complex formation was significantly increased compared to controls (612 pM vs. 121 pM; P < 0.001), and was attenuated upon inhibition of PKa. Conclusions - We demonstrate that, activation of FXII leads to thrombin generation via FIX activation by plasma kallikrein in the absence of FXI. In addition, the activation of FVIII by thrombin is essential for the FXI bypass route towards FIX activation. These findings support the idea that FXI and FIX have distinctly different roles within the intrinsic coagulation cascade upon FXII activation and may contribute to variations in bleeding in FXI-deficient subjects due to different prekallikrein plasma levels. Disclosures Visser: Bayer AG: Employment. Laux:Bayer AG: Employment. Heitmeier:Bayer AG: Employment.

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The Concentration of Exogenously Added Lipids and Endogenously Available Microparticles Are Major Determinants of Thrombin Generation in Plasma.

Blood ◽

10.1182/blood.v108.11.4000.4000 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4000-4000

Author(s):

C. Kluft ◽

P. Meijer ◽

R. Kret ◽

V. Kaufmann ◽

J. Mager

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Area Under The Curve ◽

Coagulation Factors ◽

Factor Ix ◽

Fixed Amount ◽

High Lipid ◽

Factor Ii ◽

The Difference

Abstract Thrombin generation tests are started by adding coagulation activator (e.g. tissue factor or contact activator) together with lipids. We evaluated the role of lipids in tests started with a fixed amount of tissue factor (7.16 pM) and addition of either 3.2 (high) or 0.32 (low) μM of lipids (Technothrombin ® TGA assays from Technoclone, Vienna) in both normal plasma and plasma that was ultracentrifuged (30 minutes at 15,000 g) to remove microparticles (MPs). The tests were performed in plasma samples of groups of apparently healthy individuals. It was observed in 54 healthy volunteers that starting with high or low lipids substantially influenced the total amount of thrombin generated expressed by the area under the curve (AUC) (AUC median 2492, IQR 716 versus AUC median 1154, IQR 652 nM*min, respectively), the rate of thrombin formation or velocity index (VI) (median 53.4, IQR 43.6 and median 11.7, IQR 10.6 nM/min, respectively), and the lag time to the start of thrombin generation (median 10.3, IQR 2.4 versus median 17.5, IQR 5.0 minutes, respectively). It can be concluded that the VI is the most sensitive variable showing approximately a factor of 5 difference between high and low lipid. The difference of adding high or low lipid on VI was primarily dependent upon the lipid concentration and to a limited extend influenced (univariate) by factor II levels (12%) and factor IX levels (10 %), taking into account practically all known coagulation factors (fibrinogen, II, V, VII, VIII, IX, X, XI, XII, PC, PS, PZ, TFPI, PCI) determined in the 54 plasma’s as potential determinant. In plasma of 36 volunteers microparticles were removed and VI dropped to 19% in comparison to the untreated plasma when tested with the addition of high lipid and to 3.1% with the addition of low lipid. Re-addition of MPs to a specific plasma restored VI dose dependently with an optimum at 2x104 MPs/ml. The same level of VI (63.8 and 62.0 nM/min, respectively) was reached with high and low lipid addition when 2.104 MPs/ml were added. It is concluded that endogenous MPs play an important role in thrombin generation tests, in particular but not exclusively when the test is performed with low levels of added lipids.

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In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽

10.1182/blood-2020-139712 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

Dougald Monroe ◽

Mirella Ezban ◽

Maureane Hoffman

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Plasma Levels ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor Ix ◽

Factor X ◽

Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

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Role of ozone in SOA formation from alkane photooxidation

Atmospheric Chemistry and Physics Discussions ◽

10.5194/acpd-13-24713-2013 ◽

2013 ◽

Vol 13 (9) ◽

pp. 24713-24754

Author(s):

X. Zhang ◽

R. H. Schwantes ◽

M. M. Coggon ◽

C. L. Loza ◽

K. A. Schilling ◽

...

Keyword(s):

Gas Phase ◽

Reaction Pathway ◽

Aerosol Composition ◽

Alkoxy Radicals ◽

The Difference ◽

Heterogeneous Formation ◽

Phase Chemistry ◽

Long Chain ◽

Oxygenated Products

Abstract. Long-chain alkanes, which can be categorized as intermediate volatile organic compounds (IVOCs), are an important source of secondary organic aerosol (SOA). Mechanisms for the gas-phase OH-initiated oxidation of long-chain alkanes have been well documented; particle-phase chemistry, however, has received less attention. The δ-hydroxycarbonyl, which is generated from the isomerization of alkoxy radicals, can undergo heterogeneous cyclization to form substituted dihydrofuran. Due to the presence of C=C bonds, the substituted dihydrofuran is predicted to be highly reactive with OH, and even more so with O3 and NO3, thus opening a reaction pathway that is not usually accessible to alkanes. This work focuses on the role of substituted dihydrofuran formation and its subsequent reaction with OH, and more importantly ozone, in SOA formation from the photooxidation of long-chain alkanes. Experiments were carried out in the Caltech Environmental Chamber using dodecane as a representative alkane to investigate the difference in aerosol composition generated from "OH-oxidation dominating" vs. "ozonolysis dominating" environments. A detailed mechanism incorporating the specific gas-phase photochemistry, together with the heterogeneous formation of substituted dihydrofuran and its subsequent gas-phase OH/O3 oxidation, is presented to evaluate the importance of this reaction channel in the dodecane SOA formation. We conclude that: (1) the formation of δ-hydroxycarbonyl and its subsequent heterogeneous conversion to substituted dihydrofuran is significant in the presence of NOx; (2) the ozonolysis of substituted dihydrofuran dominates over the OH-initiated oxidation under conditions prevalent in urban and rural air; and (3) a spectrum of highly-oxygenated products with carboxylic acid, ester, and ether functional groups are produced from the substituted dihydrofuran chemistry, thereby affecting the average oxidation state of the SOA.

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Effects of Plasma Kallikrein Specific Inhibitor and Active-site Blocked Factor VIIa on the Pulmonary Vascular Injury Induced by Endotoxin in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657716 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1209-1214 ◽

Cited By ~ 8

Author(s):

Mitsuhiro Uchiba ◽

Kenji Okajima ◽

Kazunori Murakami ◽

Hiroaki Okabe ◽

Shosuke Okamoto ◽

...

Keyword(s):

Vascular Injury ◽

Active Site ◽

Factor Viia ◽

Specific Inhibitor ◽

Coagulation System ◽

Plasma Kallikrein ◽

Extrinsic Pathway ◽

E Coli ◽

Intravascular Coagulation

SummaryThe acute respiratory distress syndrome (ARDS) is a serious complication of sepsis. To evaluate the role of the coagulation system in the pathogenesis of ARDS in sepsis, we examined the effects of the administration of a synthetic plasma kallikrein specific inhibitor (PKSI) and of active-site blocked factor VIIa (DEGR-VIIa) on the pulmonary vascular injury induced by E. coli endotoxin (ET) in rats. Administration of PKSI prevented the pulmonary vascular injury induced by ET as well as pulmonary histological changes in animals administered ET, but it did not affect the intravascular coagulation. The opposite effect was seen with DEGR-VIIa, which prevented the intravascular coagulation but not the pulmonary vascular injury. PKSI did not inhibit the activation of the complement system induced by ET leading to the activation of neutrophils.Findings suggest that PKSI may prevent the pulmonary vascular injury induced by ET by inhibiting kallikrein, which activates the neutrophils. The intrinsic pathway of coagulation may be more important than the extrinsic pathway in the pulmonary vascular injury produced byET.

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The Role of Factor IX in Tissue Thromboplastin Induced Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657215 ◽

1982 ◽

Vol 48 (01) ◽

pp. 054-058 ◽

Cited By ~ 9

Author(s):

A M H P van den Besselaar ◽

I E Ram ◽

G H J Alderkamp ◽

R M Bertina

Keyword(s):

Human Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor Xii ◽

Factor Xi ◽

Proteolytic Activation ◽

Human Factor Ix ◽

Tissue Thromboplastin ◽

The Difference

SummaryTissue thromboplastin apoprotein was partially purified from human brain. The apoprotein was recombined with mixed phospholipids to yield active thromboplastin. The recombined thromboplastin induced proteolytic activation of isolated human factor IX in the presence of factor VII and Ca2+. The clotting times of various deficient plasmas were determined as a function of apoprotein concentration, keeping the phospholipid concentration constant. The clotting times of a factor XII-deficient plasma were the same as those of a factor XII/factor IX-deficient plasma, except at very low apoprotein concentrations. However, under those conditions the difference in clotting times was independent of the presence of anti-factor VII serum. Similar observations were made for factor XI-deficient plasma in comparison with factor XI/factor IX-deficient plasma. These results indicate that activation of factor IX by factor VII/tissue thromboplastin does not significantly contribute to plasma coagulation.

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Evidence that Fitzgerald factor counteracts inhibition by kaolin or ellagic acid of the amidolytic properties of a plasma kallikrein

Blood ◽

10.1182/blood.v47.2.243.bloodjournal472243 ◽

1976 ◽

Vol 47 (2) ◽

pp. 243-251

Author(s):

OD Ratnoff ◽

H Saito

Keyword(s):

Cytochrome C ◽

Ellagic Acid ◽

Factor Vii ◽

Plasma Kallikrein ◽

High Molecular Weight Kininogen ◽

Intrinsic Pathway ◽

Fletcher Factor ◽

The Relationship ◽

Plasma Reactions

Fitzgerald trait, an asymptomatic disorder, is associated with abnormalities of surface-mediated plasma reactions, including coagulation via the intrinsic pathway, augmentation of the clot- promoting properties of factor VII, kaolin-mediated fibrinolysis, kinin generation, and enhancement of vascular permeability by diluted plasma (PF/Dil). These abnormalities can be corrected by Fitzgerald factor, an agent probably identical with high molecular weight kininogen found in normal, but not Fitzgerald-trait plasma. Our preparations of Fitzgerald factor possessed a second property. Amidolysis of alpha-N-benzoyl-L- proline-L-phenylalanine-L-arginine-pnitroanilide by a plasma kallikrein (activated Fletcher factor) was inhibited by kaolin or solutions of ellagic acid. Addition of preparations of Fitzgerald factor to kaolin or to solutions of ellagic acid counteracted their inhibitory properties. The action of these preparations was duplicated by solutions of cytochrome C or IgG, suggesting that these agents may inhibit the negative charges of kaolin or ellagic acid. Fitzgerald factor enhanced amidolysis of both normal and Fitzgerald-trait plasmas exposed to kaolin, effects not duplicated by cytochrome C or IgG. Whether or not the two properties of our preparations of Fitzgerald factor are related to the same agent is not yet certain. The relationship between these observations and the biologic role of Fitzgerald factor remains to be investigated.

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Cold Promoted Activation of Factor VII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649054 ◽

1972 ◽

Vol 28 (02) ◽

pp. 182-193 ◽

Cited By ~ 18

Author(s):

H Gjønniess

Keyword(s):

Ellagic Acid ◽

Factor Vii ◽

Coagulation System ◽

Plasma Kallikrein ◽

Glandular Kallikrein ◽

Kallikrein Activity

SummarySeveral known activators of the plasma kallikrein system were tested for the ability to induce cold promoted activation of factor VII.All the prekallikrein activators tested (EACA, trypsin, acetone, urokinase, kaolin, Liquoid, ellagic acid) revealed generation of factor VII activator activity (CPA). Simultaneously the plasma kallikrein system was activated. A good correlation was demonstrated between the factor VII activation and the kallikrein activity.Glandular kallikrein, bradykinin, or kallidin revealed no effect on the coagulation system. Thus, the correlation between the factor VII activation and the kallikrein system seemed to be restricted to plasma kallikrein.

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