Functional Characterization of a Variant Factor XII (F XII Locarno) in a Cross Reacting Material Positive F XII Deficient Plasma

1992 ◽  
Vol 67 (02) ◽  
pp. 219-225 ◽  
Author(s):  
Walter A Wuillemin ◽  
Miha Furlan ◽  
Hans Stricker ◽  
Bernhard Lämmle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Single Chain ◽  
Sulfate Adsorption ◽  
Prolonged Incubation ◽  
Functional Studies

SummaryThe plasma of a healthy woman was found to contain half normal factor XII (FXII) antigen level (0.46 U/ml) without any FXII clotting activity (<0.01 U/ml). The variant FXII in this plasma, denoted as FXII Locarno, was partially characterized by immunological and functional studies on the proposita’s plasma. FXII Locarno is a single chain molecule with the same size (M r = 80 kDa) as normal FXII. Isoelectric focusing suggested an excess of negative charge in the variant FXII as compared to normal FXII. In contrast to FXII in normal plasma, FXII Locarno was not proteolytically cleaved upon prolonged incubation of proposita’s plasma with dextran sulfate. Adsorption to kaolin was similar for both, abnormal and normal FXII. Incubation of the proposita’s plasma with dextran sulfate and exogenous plasma kallikrein showed normal cleavage of FXII Locarno outside of the tentative disulfide loop Cys340-Cys467, but only partial cleavage within this disulfide loop. Furthermore, plasma kallikrein-cleaved abnormal FXII showed neither amidolytic activity nor proteolytic activity against factor XI and plasma prekallikrein.These results suggest a structural alteration of FXII Locarno, affecting the plasma kallikrein cleavage site Arg353-Val354 and thus formation of activated FXII (a-FXIIa).

Functional characterization of an abnormal factor XII molecule (F XII Bern)

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 997-1004
Author(s):  
WA Wuillemin ◽  
I Huber ◽  
M Furlan ◽  
B Lammle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Molecular Defect ◽  
Factor Xii ◽  
Single Chain ◽  
Functional Studies ◽  
Functional Defect

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

scholarly journals Functional characterization of an abnormal factor XII molecule (F XII Bern)

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 997-1004 ◽  
Author(s):  
WA Wuillemin ◽  
I Huber ◽  
M Furlan ◽  
B Lammle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Molecular Defect ◽  
Factor Xii ◽  
Single Chain ◽  
Functional Studies ◽  
Functional Defect

Abstract An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

scholarly journals Plasminogen activators in dextran sulfate-activated euglobulin fractions: a molecular analysis of factor XII- and prekallikrein- dependent fibrinolysis

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 994-999
Author(s):  
J Hauert ◽  
G Nicoloso ◽  
WD Schleuning ◽  
F Bachmann ◽  
M Schapira
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Dextran Sulfate ◽  
Plasminogen Activators ◽  
Contact System ◽  
Tissue Type ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Single Chain ◽  
Type Plasminogen Activator

To elucidate the mechanism by which activation of the contact system of blood coagulation leads to expression of fibrinolytic activity, we have determined the molecular characteristics of the plasminogen activators present in dextran sulfate-treated euglobulin fractions by electrophoretic-zymographic analysis and specific immunoadsorption. In addition to free and protease inhibitor-bound tissue-type plasminogen activator (t-PA), dextran sulfate precipitates of euglobulins contained the complex formed between plasma kallikrein and C1-inhibitor, an indicator of prekallikrein activation. These precipitates also contained substantial fibrinolytic activity related to urinary-type plasminogen activator (u-PA). Autoradiographic analysis was then used to evaluate the cleavage of 125I-single-chain u-PA (prourokinase) in dextran sulfate euglobulins as well as after exposure to kallikrein or beta-factor XIIa. This analysis supported the conclusion that plasma kallikrein-mediated cleavage and activation of single-chain u-PA is the mechanism operative for the development of lytic activity in euglobulin precipitates following activation of the contact system.

Functional Characterization of a Variant Prekallikrein (PK Zürich)

1993 ◽  
Vol 70 (03) ◽  
pp. 427-432 ◽  
Author(s):  
W A Wuillemin ◽  
M Furlan ◽  
A von Felten ◽  
B Lämmle
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
High Molecular Weight ◽  
Dextran Sulfate ◽  
Functional Characterization ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Normal Plasma ◽  
Sulfate Activation

SummaryThe plasma of a 68-year-old man with cross reacting material (CRM)-positive prekallikrein (PK) deficiency was studied. PK clotting activity was <0.01 U/ml, and PK antigen was 0.1 U/ml. No circulating anticoagulant against PK was detectable. The abnormal PK molecule, denoted as prekallikrein Zürich, was partially characterized by immunological and functional studies on the propositus’ plasma. Immunobiotting analysis showed the abnormal PK being a single chain molecule of the same M r (80 kDa) as normal PK. Dextran sulfate activation of the propositus’ plasma did not lead to proteolytic cleavage of the variant PK molecule, in contrast to dextran sulfate activation of a mixture of 1 volume normal plasma and 9 volumes CRM-negative PK deficient plasma. Agarose gel electrophoresis followed by immunoblotting demonstrated that PK Zürich was complexed with high molecular weight kininogen similarly to PK in normal plasma. Incubation of the propositus’ plasma with purified β-FXIIa resulted in impaired cleavage of PK Zürich when compared with PK hydrolysis in a mixture of 10% normal plasma and 90% CRM-negative PK deficient plasma. Moreover, proteolytically cleaved PK Zürich showed no enzymatic activity against factor XII and high molecular weight kininogen.These studies show that the functional defect of prekallikrein Zürich is due to an impaired cleavage by activated factor XII and probably the lack of enzymatic activity of the cleaved variant molecule.

scholarly journals Plasminogen activators in dextran sulfate-activated euglobulin fractions: a molecular analysis of factor XII- and prekallikrein- dependent fibrinolysis

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 994-999 ◽  
Author(s):  
J Hauert ◽  
G Nicoloso ◽  
WD Schleuning ◽  
F Bachmann ◽  
M Schapira
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Dextran Sulfate ◽  
Plasminogen Activators ◽  
Contact System ◽  
Tissue Type ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Single Chain ◽  
Type Plasminogen Activator

Abstract To elucidate the mechanism by which activation of the contact system of blood coagulation leads to expression of fibrinolytic activity, we have determined the molecular characteristics of the plasminogen activators present in dextran sulfate-treated euglobulin fractions by electrophoretic-zymographic analysis and specific immunoadsorption. In addition to free and protease inhibitor-bound tissue-type plasminogen activator (t-PA), dextran sulfate precipitates of euglobulins contained the complex formed between plasma kallikrein and C1-inhibitor, an indicator of prekallikrein activation. These precipitates also contained substantial fibrinolytic activity related to urinary-type plasminogen activator (u-PA). Autoradiographic analysis was then used to evaluate the cleavage of 125I-single-chain u-PA (prourokinase) in dextran sulfate euglobulins as well as after exposure to kallikrein or beta-factor XIIa. This analysis supported the conclusion that plasma kallikrein-mediated cleavage and activation of single-chain u-PA is the mechanism operative for the development of lytic activity in euglobulin precipitates following activation of the contact system.

Analysis of Intrinsic Fibrinolysis in Human Plasma Induced by Dextran Sulfate

1992 ◽  
Vol 67 (04) ◽  
pp. 440-444 ◽  
Author(s):  
Hiroko Tsuda ◽  
Toshiyuki Miyata ◽  
Sadaaki Iwanaga ◽  
Tetsuro Yamamoto
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Dextran Sulfate ◽  
Tissue Type ◽  
Factor Xii ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Major Pathway ◽  
Normal Human

SummaryThe analysis of normal human plasma by fibrin autography revealed four species of plasminogen activator (PA) activity related to tissue-type PA, factor XII, prekallikrein and urokinase-type PA (u-PA). The u-PA activity increased significantly by incubating plasma with dextran sulfate. This increase was coincident with both the cleavage of factor XII and the complex formation of activated factor XII with its plasma inhibitors, which were determined by immunoblotting procedure. The dextran sulfate-dependent activation of u-PA required both factor XII and prekallikrein, but did not require either plasminogen or factor XI. High molecular weight kininogen was required only at a low concentration of dextran sulfate. Thus the results indicate that the factor XII and prekallikrein-mediated activation of single chain u-PA (scu-PA) operates as a major pathway of scu-PA activation in whole plasma in contact with dextran sulfate.

scholarly journals The contact activation mechanism in human plasma: activation induced by dextran sulfate

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1225-1233 ◽  
Author(s):  
F van der Graaf ◽  
FJ Keus ◽  
RA Vlooswijk ◽  
BN Bouma
Keyword(s):  
Human Plasma ◽  
Dextran Sulfate ◽  
Activation Mechanism ◽  
Factor Xii ◽  
Contact Activation ◽  
Prolonged Incubation ◽  
Normal Plasma ◽  
Essential Components ◽  
Factor Xiia ◽  
Kallikrein Activity

Abstract Incubation of normal human plasma with dextran sulfate for 7 min at 4 degrees C generates kallikrein amidolytic activity. No kallikrein activity is generated in factor XII or prekallikrein-deficient plasma and only small amounts (8%) in high molecular weight (HMW) kininogen- deficient plasma. Addition of specific antisera directed against prekallikrein or HMW kininogen to normal plasma blocked the generation of kallikrein activity by dextran sulfate. Thus, factor XII, prekallikrein, and HMW kininogen are essential components for optimal activation of prekallikrein. The role of limited proteolysis in the activation of prekallikrein induced by dextran sulfate was studied by adding 125I-prekallikrein to plasma. The generation of kallikrein activity paralleled the proteolytic cleavage of prekallikrein as judged on SDS gels in the presence of reducing agents. The same cleavage fragments were observed as obtained by activation of purified prekallikrein by beta-factor-XIIa. Addition of 131I-HMW kininogen and 125I-factor XII or 131I-HMW kininogen and 125I-prekallikrein to normal plasma followed by activation with dextran sulfate and analysis on SDS gels indicated that the observed cleavage of prekallikrein and HMW kininogen is fast compared to the observed cleavage of factor XII, which is much slower and less extensive. During the first minutes of incubation of normal plasma with dextran sulfate, mainly alpha-factor- XIIa is formed. During prolonged incubation, beta-factor-XIIa is also formed.

Role Of Hmw Kininogen In Polysaccharide Sulfate-Mediated Activation Of Factor XII

1981 ◽  
Author(s):  
H Kato ◽  
T Shimada ◽  
T Sugo ◽  
S Iwanaga
Keyword(s):  
Sulfur Content ◽  
Light Chain ◽  
Dextran Sulfate ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Charged Surfaces ◽  
Fluorogenic Peptide Substrate ◽  
Derivatives Of ◽  
Fluorogenic Peptide

We have shown that bovine HMW kininogen (HMW KGN) accelerates the activation of Factor XII (XII) and prekallikrein (Prek) in the presence of kaolin, adsorbing on kaolin through fragment 1.2 region and forming a complex with Prek through light chain region. The present study was undertaken to examine the functional role of HMW KGN in the activation of XII and Prek with other negatively-charged surfaces than kaolin. XII was incubated with a variety of substances, HMW KGN and Prek, and plasma kallikrein generated was estimated using a fluorogenic peptide substrate, Z- Phe-Arg-MCA.Among 8 kinds of polysaccharide sulfates (dextran sulfate, xylan sulfate, amylopectin sulfate, chondroitin polysulfate, heparan sulfate, amylose sulfate, heparin and carrageenin), amylose sulfate had the most potent ability to activate XII. The experiments using fourteen amylose sulfates with different sulfur content and different viscosity revealed that some amylose sulfates with a higher sulfur content (18%) activated strikingly XII and Prek in the presence of HMW KGN. In the reaction, fragment 1.2-light chain region in HMW KGN was shown to be essential, using the various derivatives of HMW KGN. The rate of XII activation was quite dependent on the concentration of HMW KGN and amylose sulfate. At higher concentrations, both of them inhibited the activation reaction. On the other hand, sulfatide has been shown to accelerate the activation of XII with plasma kallikrein by Fujikawa et al. and the reaction was inhibited by the addition of HMW KGN. In the present study, the activation of XII and Prek with sulfatides as estimated by the generation of kallikrein, was accelerated by HMW KGN. This reaction was also dependent on the concentrations of sulfatides and HMW KGN, and fragment 1.2-light chain region in HMW KGN was shown to be essential.These results indicate that HMW KGN is required to accelerate the activation of XII and Prek with polysaccharide sulfate and sulfatide as well as kaolin.

Outside-In Signaling upon Assembly on Endothelial Cells of the Proteins of the Plasma Kallikrein/Kinin System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1024-1024
Author(s):  
Fakhri Mahdi ◽  
Zia Shariat-Madar ◽  
Alvin Schmaier
Keyword(s):  
Endothelial Cells ◽  
Zinc Ion ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Receptor Complex ◽  
Single Chain ◽  
Potential Growth ◽  
Receptor System ◽  
Kinin System ◽  
Kallikrein Kinin System

Abstract Investigations from our laboratory have shown that there is a multiprotein receptor system on endothelial cells (HUVEC) consisting of gC1qR, uPAR, and cytokeratin 1 for high molecular weight kininogen (HK) and factor XII (XII) (Blood97:2342; 99:3585) that serves as a presentation receptor for prekallikrein (PK) activation. When PK binds to HK on HUVEC, it is activated to plasma kallikrein by the serine protease prolylcarboxypeptidase (JBC277:17962; Blood103:4554). HK is also known to have anti-proliferative and anti-angiogenic activity. We asked if there is outside-in signaling through this HUVEC receptor complex. Initial investigations determined if single chain urokinase (ScuPA) or XII stimulates Erk 1 and 2 (MAPK42 and 44) (MAPK) in HUVEC. Independently ScuPA (5–200 nM) or XII (15–200 nM) in the presence of 0.05 mM zinc ion stimulates HUVEC MAPK expression and it is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Two chain uPA or APMSF-treated ScuPA upregulates MAPK to the same extent as ScuPA. Similarly, XIIa or APMSF-treated XII upregulates MAPK like XII. Since HK, ScuPA, XII and vitronectin (VN) all bind to the same region on uPAR (JBC279:16621), studies focused on the role of uPAR in these activities. Mab3B10 to the HK, ScuPA, XII, and VN binding site on uPAR’s Domain 2 (D2) blocks ScuPA or XII upregulation of MAPK. Peptides LRG20, YLP20, PGS20, or FHN20 from uPAR’s D2 (L166-N200) block ScuPA or XII upregulation of MAPK. Similarly, HKa (1 micromolar) and peptides from the HK Domain 5 (G469-H498) HUVEC binding region (GGH18, HKH20) (JBC270:19256) block ScuPA- or XII-induced MAPK expression. Treatment of HUVEC with 5-20 mM methyl-beta-cyclodextran (MBCD) or fillipin (1 microgram/ml), agents known to disrupt lipid rafts, do not interfere with ScuPA- or XII-induced MAPK upregulation. These combined data indicate that zymogen ScuPA or XII, two proteins with growth factor regions, directly signal through uPAR to upregulate MAPK to possibly induce cellular proliferative activities. HK or its activated fragments bound to uPAR inhibits these potential growth promoting activities. These studies imply that HKa’s anti-proliferative and anti-angiogenic activities are mediated through its interaction with uPAR. These investigations also indicate that, in addition to a presentation complex for PK activation, the HUVEC multiprotein receptor complex for HK, XII, and ScuPA has an auto-regulating outside-in signaling system in endothelials cells.

Effect Of Two Soluble "Activators" On The Activity Of Factor XII And Factor XIIa (Hageman Factor)

1981 ◽  
Author(s):  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Dextran Sulfate ◽  
Maximal Activity ◽  
Contact System ◽  
Factor Xii ◽  
Single Chain ◽  
Amidolytic Activity ◽  
Factor Xiia ◽  
Procoagulant Effect ◽  
Dose Dependent ◽  
Chain Form

Certain activators of the contact system of coagulation have been reported to induce full activity in the singlechain form (80,000 MW) of Factor XII (FXII). The effects of soluble ellagic acid (EA) and dextran sulfate (DXS) (∼500,000 MW) on purified components of the contact system were studied. Soluble EA (<40 yM) was found to exert a dose-dependent procoagulant effect on purified FXII added to FXII-deficient plasma although the maximal activity observed was much less than that elicited by kaolin in the same mixtures. 5 μM soluble EA increased the amidolytic activity of FXII against H-D-Pro-Phe-Arg-pNA from 2.5 to 5.0 mol substrate/mol enzyme/min. Purified β-FXIIa (28,000 MW) or FXII that had been preincubated with trypsin hydrolyzed 900 mol substrate/mol enzyme/min in the presence or absence of soluble EA. Thus, soluble EA induces minimal (≤0.3%) amidolytic activity in FXII. We also examined the generation of FXIa in the presence of 5 μM soluble EA, FXI, high MW kininogen, and FXII or α-FXIIa (two-chain form, 80.000 MW). The rate of cleavage of 125I-FXI in the presence of 10 μg/ml DXS, FXI, high MW kininogen, and FXII or α-FXIIa was also measured. In the presence of either DXS or soluble EA under the concentrations and conditions employed, α-FXIIa exhibited an initial rate of FXI activation that was 20 times higher than that achieved by single-chain FXII. In addition, the presence of both high MW kininogen and either EA or DXS enhanced the activity of α-FXIIa 20 fold in the activation of FXI. The results show that single chain FXII in the presence of either DXS or soluble EA expresses less than 5% of the enzymatic activities of its proteolytically derived forms whereas either “activator” greatly enhances the action of α-FXIIa on FXI in the presence of high MW kininogen.

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