The Fibronectin Type II Domain of Factor XII Ensures Zymogen Quiescence

AbstractFactor XII (FXII) zymogen activation requires cleavage after arginine 353 located in the activation loop. This cleavage can be executed by activated FXII (autoactivation), plasma kallikrein (PKa), or plasmin. Previous studies proposed that the activation loop of FXII is shielded to regulate FXII activation and subsequent contact activation. In this study, we aimed to elucidate this mechanism by expressing and characterizing seven consecutive N-terminally truncated FXII variants as well as full-length wild-type (WT) FXII. As soon as the fibronectin type II domain is lacking (FXII Δ1–71), FXII cleavage products appear on Western blot. These fragments display spontaneous amidolytic activity, indicating that FXII without the fibronectin type II domain is susceptible to autoactivation. Additionally, truncated FXII Δ1–71 is more easily activated by PKa or plasmin than full-length WT FXII. To exclude a contribution of autoactivation, we expressed active-site incapacitated FXII truncation variants (S544A). FXII S544A Δ1–71 is highly susceptible to cleavage by PKa, indicating exposure of the activation loop. In surface binding experiments, we found that the fibronectin type II domain is non-essential for binding to kaolin or polyphosphate, whereas the following epidermal growth factor-like domain is indispensable. Binding of full-length FXII S544A to kaolin or polyphosphate increases its susceptibility to cleavage by PKa. Moreover, the activation of full-length WT FXII by PKa increases approximately threefold in the presence of kaolin. Deletion of the fibronectin type II domain eliminates this effect. Combined, these findings suggest that the fibronectin type II domain shields the activation loop of FXII, ensuring zymogen quiescence.

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Properties of sulfatides in factor-XII-dependent contact activation

Blood ◽

10.1182/blood.v59.1.69.69 ◽

1982 ◽

Vol 59 (1) ◽

pp. 69-75 ◽

Cited By ~ 41

Author(s):

G Tans ◽

JH Griffin

Keyword(s):

Normal Values ◽

Limited Proteolysis ◽

Factor Xii ◽

Contact Activation ◽

Amidolytic Activity ◽

Prolonged Incubation ◽

Coagulant Activity ◽

Plasma Factor ◽

Normal Human ◽

Kallikrein Activity

Abstract Incubation of normal human plasma with low amounts of sulfatides resulted in the initiation of intrinsic coagulation and the appearance of kallikrein activity. The optimal initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of factor XII, high molecular weight kininogen, and prekallikrein. Since the activated partial thromboplastin clotting times in prekallikrein- deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon of autocorrection was studied and found to be even more pronounced in the presence of sulfatides. Autocorrection was essentially completed in 5 min in the presence of sulfatides, whereas a preincubation of 15–20 min was required in the presence of kaolin. The limited proteolysis of 125I-factor XII in plasma during incubation with activating material or during clotting was determined. Cleavage of factor XII was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. In prekallikrein-deficient plasma, factor XII cleavage was completed within 5 min in the presence of sulfatides and within 15 min in the presence of kaolin. Thus, the appearance of factor-XII-dependent coagulant activity correlates with the limited proteolysis of factor XII when normal or prekallikrein- deficient plasma is activated by sulfatides or kaolin.

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Molecular Mechanisms of Contact Activation

10.1055/s-0039-1682647 ◽

1977 ◽

Author(s):

John H. Griffin

Keyword(s):

Molecular Mechanisms ◽

Factor Xii ◽

Contact Activation ◽

Surface Binding ◽

Factor Xi ◽

Proteolytic Activation ◽

Charged Surfaces ◽

Fletcher Factor ◽

New Hypothesis ◽

Rate Of Activation

Exposure of human plasma to various negatively charged surfaces (glass, kaolin, connective tissue components, urate crystals, endotoxin, etc.) leads to ‘contact activation”of plasma and thereby initiates the activation of the intrinsic coagulation, the kinin-forming, and the plasma fibrinolytic systems. Studies of plasmas which are functionally defective in contact activation reactions implicate the following proteins in contact activation reactions: (1) Hageman Factor (HF) (Factor XII); (2) prekallikrein (Fletcher Factor); (3) high MW kininogen (Fitzgerald, Flau¡eac, or Williams Factor) (Contact Activation Cofactor); and (4) Factor XI. Recent studies suggest that a previously postulated HF-dependent plasminogen proactivator is identical to prekallikrein.A mixture of purified HF, high MW kininogen, prekallikrein, and kaolin gives the same rapid rate of activation of purified Factor XI as does an equivalent aliquot of plasma plus kaolin, thus suggesting that contact activation of Factor XI is fully explicable in terms of the interactions between these proteins. Using these purified proteins, the molecular mechanisms of contact activation have been studied extensively, and the results which will be presented support the following new hypothesis. Surface-binding of HF does not convert a detectable fraction (<1%) of HF molecules to active enzymes as previously supposed, but instead it renders HF much more susceptible (100 to 400-fold) to proteolytic activation by plasmin or kallikrein in the presence of high MW kininogen. High MW kininogen is a surface-bound HF cofactor for each step in the reciprocal activation of prekallikrein and HF, and for the limited cleavage of Factor XI by activated HF.

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FACTOR XII, PLASMA PREKALLIKREIN ,α2-MACROGLOBULIN AND C1-INHIBITOR LEVELS IN RENAL ALLOGRAFT RECIPIENTS DURING IMMUNOSUPPRESSION WITH CYCLOSPORIN A

10.1055/s-0038-1644122 ◽

1987 ◽

Author(s):

A Lohri ◽

B Huser ◽

B Lämmle ◽

M Oberholzer ◽

G Thiel ◽

...

Keyword(s):

Cyclosporin A ◽

Renal Allograft ◽

Serum Bilirubin ◽

Acute Phase Reactant ◽

Endothelial Damage ◽

Factor Xii ◽

Contact Activation ◽

Amidolytic Activity ◽

Phase Reactant ◽

Α2 Macroglobulin

Factor XII clotting activity (FXII), Plasma Pre-kallikrein amidolytic activity (PK), 2-Macroglobulin ( α2-M) and cT-Inhibitor (cT-Inh) antigen have been measured in 17 patients immediately before and sequentially up to four months after kidney transplantation. Based on suspected Cyclosporin A (CyA) induced endothelial damage, activation of the contact system with resulting consumption of the contact activation factors was^evaluated. Before transplantation, FXII, PK, α2-M, Cl-Inh levels were 99±27%, 102±21%, 115±55%, and 129±32%, respectively. In the first two weeks after transplantation FXII decreased to 65±27%, PK to 67±20% and α2-M to 88±42%; Cl-Inh rose to a maximum of 201±44% (mean ± S. D.)(2p<0.001). Mean FXII levels correlated positively with PK, α2-M and albumin and negatively with CyA level and dose and serum bilirubin. PK and α2-M correlated positively with each other and with albumin and negatively with creatinine, bilirubin and CyA (p<0.01). The changes of FXII, PK and α2-M after transplantation suggest an influence of CyA on production or consumption of these factors. The behaviour of the cT-Inh may be unspecific and related to its action of an acute phase reactant.

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Identification of a Putative Binding Site for Negatively Charged Surfaces in the Fibronectin Type II Domain of Human Factor XII

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614171 ◽

2000 ◽

Vol 84 (12) ◽

pp. 1057-1065 ◽

Cited By ~ 21

Author(s):

Henk te Velthuis ◽

Manuela Helmer-Citterich ◽

C. Hack ◽

Franca Citarella

Keyword(s):

Amino Acid ◽

Binding Site ◽

Three Dimensional ◽

Coagulation Factor ◽

Factor Xii ◽

Type Ii ◽

Three Dimensional Model ◽

Functional Sites ◽

Charged Surfaces ◽

Fibronectin Type

SummaryMonoclonal antibodies directed against functional sites of proteins provide useful tools for structure-function studies. Here we describe a mAb, KOK5, directed against the heavy chain region of human coagulation factor XII (FXII), which inhibits kaolin-induced clotting activity by preventing the binding of FXII to kaolin. Furthermore, mAb KOK5 enhances FXII susceptibility for cleavage by kallikrein and supports FXII autoactivation. Hence, mAb KOK5 likely is directed against the binding site of FXII for negatively charged surfaces. Screening of two phage-displayed random peptide libraries with mAb KOK5 selected phages that could be grouped on the basis of two amino acid consensus sequences: A) FXFQTPXW and B) HQ/LCTHR/KKC. Sequence A contains two motifs: one shares homology with FXII amino acid residues 30-33 (FPFQ), the second one with residues 57-60 (TPNF); both amino acid stretches belonging to the fibronectin type II domain of FXII. Sequence B also reveals homology with part of the fibronectin type II domain, i.e. the stretch 40-47 (HKCTHKGR). A three-dimensional model of FXII residues 28-65, obtained by homology modeling, indicated that the three amino acid stretches 30-33, 40-47 and 57-60 are close to each other and accessible for the solvent, i.e. in a form available for interaction with the monoclonal antibody, suggesting that mAb KOK5 recognizes a discontinuous epitope on the fibronectin type II domain of FXII. Peptides corresponding to FXII sequences 29-37 (FXII29-37) or 39-47 (FXII39-47), were synthesized and tested for the capability to inhibit FXII binding to negatively charged surfaces. Peptide FXII39-47 inhibited the binding of labeled FXII to kaolin and effectively prevented both dextran sulfate-and kaolin-induced activation of the contact system in plasma. Hence, we suggest that the fibronectin type II domain of FXII, in particular residues 39 to 47, contribute to the binding site of FXII for negatively charged surfaces.

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Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

Nature Communications ◽

10.1038/s41467-021-25888-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Marco Heestermans ◽

Clément Naudin ◽

Reiner K. Mailer ◽

Sandra Konrath ◽

Kristin Klaetschke ◽

...

Keyword(s):

Partial Thromboplastin Time ◽

Thrombin Generation ◽

Thrombus Formation ◽

Coagulation Factors ◽

Activated Partial Thromboplastin Time ◽

Plasma Kallikrein ◽

Factor Xii ◽

Contact Activation ◽

Intrinsic Pathway ◽

Surface Binding

AbstractContact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

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Hageman Factor (HF, Factor XII) -Dependent Activation of Coagulation, Kinin-Forming, and Fibrinolytic Pathways by Surface Contact

10.1055/s-0039-1684812 ◽

1979 ◽

Author(s):

J.H. Griffin

Keyword(s):

Molecular Mechanisms ◽

Human Platelets ◽

Factor Xii ◽

Contact Activation ◽

Surface Binding ◽

Factor Xi ◽

Proteolytic Activation ◽

Hageman Factor ◽

Charged Surfaces ◽

Inactive Form

Exposure of plasmo to various negatively charged surfaces (glass, kaolin, connective tissue components, urate crystals, etc.) initiates contact activation of plasma resulting in the activation of the intrinsic coagulation, the kinin-forming, and the fibrinolytic systems. Proteins involved in these reactions include HF, Factor XI, prekallikrein, and high MW kininogen. Recent studies will be summarized that provide new information on the molecular mechanisms responsible for contact activation. Surface-binding of HF does not convert HF from an inactive form to an active enzyme, as previously supposed; rather it makes HF much more susceptible (100 to 500-fold) to proteolytic activation by kallikrein. plasmin, or other proteases. Reciprocal proteolytic activation of prekallikrein and HF occurs, and high MW kininogen is a surface-bound cofactor for each step. The potential influence of different cells on the activation of the contact system will also be discussed. Human platelets have been shown to stimulate the activation of HF and of Factor XI. Endothelial cells contain an enzyme capable of activating Hageman factor. Moreover, human basophils secrete an enzyme capable of activating HF. Thus, it appears that proteins derived from a number of cells can influence the nature and extent ot contact activation reactions.

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Properties of sulfatides in factor-XII-dependent contact activation

Blood ◽

10.1182/blood.v59.1.69.bloodjournal59169 ◽

1982 ◽

Vol 59 (1) ◽

pp. 69-75 ◽

Cited By ~ 1

Author(s):

G Tans ◽

JH Griffin

Keyword(s):

Normal Values ◽

Limited Proteolysis ◽

Factor Xii ◽

Contact Activation ◽

Amidolytic Activity ◽

Prolonged Incubation ◽

Coagulant Activity ◽

Plasma Factor ◽

Normal Human ◽

Kallikrein Activity

Incubation of normal human plasma with low amounts of sulfatides resulted in the initiation of intrinsic coagulation and the appearance of kallikrein activity. The optimal initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of factor XII, high molecular weight kininogen, and prekallikrein. Since the activated partial thromboplastin clotting times in prekallikrein- deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon of autocorrection was studied and found to be even more pronounced in the presence of sulfatides. Autocorrection was essentially completed in 5 min in the presence of sulfatides, whereas a preincubation of 15–20 min was required in the presence of kaolin. The limited proteolysis of 125I-factor XII in plasma during incubation with activating material or during clotting was determined. Cleavage of factor XII was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. In prekallikrein-deficient plasma, factor XII cleavage was completed within 5 min in the presence of sulfatides and within 15 min in the presence of kaolin. Thus, the appearance of factor-XII-dependent coagulant activity correlates with the limited proteolysis of factor XII when normal or prekallikrein- deficient plasma is activated by sulfatides or kaolin.

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Synchronized Inhibition of the Phospholipid Mediated Autoactivation of Factor XII in Plasma by β2-glycoprotein I and Anti-β2-glycoprotein I

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653871 ◽

1995 ◽

Vol 73 (05) ◽

pp. 798-804 ◽

Cited By ~ 48

Author(s):

Inger Schousboe ◽

Margit Søe Rasmussen

Keyword(s):

Lupus Erythematosus ◽

Lupus Anticoagulant ◽

Response Curve ◽

Inhibitory Effect ◽

Factor Xii ◽

High Antibody ◽

Contact Activation ◽

Maximal Inhibition ◽

Lupus Anticoagulants ◽

Glycoprotein I

SummaryLupus anticoagulants are a group of antibodies commonly found in patients with autoimmune diseases such as systemic lupus erythematosus. Lupus anticoagulants inhibit phospholipid dependent coagulation and may bind to negatively charged phospholipids. Recent studies have suggested an association between anti-β2-glycoprotein I and a lupus anticoagulant, whose activity is frequently dependent on the presence of β2-glycoprotein I. Based on these observations, the effect of anti-β2-glycoprotein I on the autoactivation of factor XII in plasma was investigated. Autoactivation initiated by the presence of negatively charged phospholipids, but not by sulfatide, was strongly inhibited by immunoaffinity purified anti-β2-glycoprotein I. The dose-response curve of anti-β2-gly coprotein I was identical with that of a precipitating antibody, showing no inhibition at low and high antibody dilutions and maximal inhibition at an intermediate dilution. At high antibody concentrations, an increased rate of factor Xlla activation was observed. This increase was of the same magnitude as the decreased rate observed in plasma supplemented with the same amount of β2-glycoprotein I as in the plasma itself. This confirms the inhibitory effect of β2-GP-I on the contact activation and shows that inhibition is effective on the autoactivation of factor XII in plasma. The inhibitory action of β2-glycoprotein I was independent of the inhibition caused by the anti- β2-glycoprotein I/β2-glycoprotein I complex suggesting a synchronized inhibition of factor XII autoactivation by β2-glycoprotein I and anti-β2-gly coprotein I. The inhibition caused by the antibody is suggested to be caused by a reduced availability of negatively charged phospholipids due to the binding of the anti-β2- GP-I/β2-GP-I complex. This complex may be a lupus anticoagulant.

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Plasma Contact Activation and Decrease of Factor V Activity on Negatively-Charged Polyelectrolytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661020 ◽

1984 ◽

Vol 51 (01) ◽

pp. 061-064 ◽

Cited By ~ 2

Author(s):

M C Boffa ◽

B Dreyer ◽

C Pusineri

Keyword(s):

Time Dependent ◽

Initial Contact ◽

Factor Xii ◽

Factor V ◽

Contact Activation ◽

Polyelectrolyte Complexes ◽

Plasma Factor ◽

Fresh Plasma ◽

Direct Adsorption

SummaryThe effect of negatively-charged polymers, used in some artificial devices, on plasma clotting and kinin systems was studied in vitro using polyelectrolyte complexes.Contact activation was observed as an immediate, transient and surface-dependent phenomenon. After incubation of the plasma with the polymer a small decrease of factor XII activity was noticed, which corresponded to a greater reduction of prekallikrein activity and to a marked kinin release. No significant decrease of factor XII, prekallikrein, HMW kininogen could be detected immunologically. Only the initial contact of the plasma with the polyelectrolyte lead to activation, subsequently the surface became inert.Beside contact activation, factor V activity also decreased in the plasma. The decrease was surface and time-dependent. It was independent of contact factor activation, and appeared to be related to the sulfonated groups of the polymer. If purified factor V was used instead of plasma factor V, inactivation was immediate and not time-dependent suggesting a direct adsorption on the surface. A second incubation of the plasma-contacted polymer with fresh plasma resulted in a further loss of Factor V activity.

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