Dextran Sulfate Stimulated Fibrinolytic Activity In Whole Human Plasma: Dependence On The Contact Activation System And On 4 Urokinase-Related Antigen

1981 ◽  
Author(s):  
Lindsey A Miles ◽  
Zuleika Rothschild ◽  
John H Griffin
Keyword(s):  
Human Plasma ◽  
Fibrinolytic Activity ◽  
Dextran Sulfate ◽  
Factor Xii ◽  
Dependent Manner ◽  
Contact Activation ◽  
Prolonged Incubation ◽  
Normal Plasma ◽  
Fibrin Plate ◽  
Activation System

The generation of fibrinolytic activity in whole human plasma in the presence of dextran sulfate was studied. Plasma was preincubated with N-flufenamyl-β-alanine to remove its antiplasmin and anti activator activities and then incubated with 50 μg/ml dextran sulfate (Mr - 500,000) for 30 min at 40. The initial fibrinolytic activity in 30 min, as assessed on a 125I-fibrin plate, was equivalent to approximately 9 ng/ml purified plasmin. A fraction of goat antibodies to plasminogen blocked the fibrinolytic activity of stimulated plasma, indicating that the activity was plasminogen dependent. Plasmas genetically deficient in either prekallikrein or Factor XII (Hageman Factor) showed a diminished initial rate of generation of fibrinolytic activity in response to dextran sulfate. However, after prolonged incubation (∼3 hr) of stimulated deficient plasmas with fibrin, the fibrinolytic activity approached that of stimulated normal plasma. When normal plasma was preincubated with the gamma fraction of goat antibodies made against purified urokinase, the dextran sulfate stimulated fibrinolytic activity was markedly decreased in a dose dependent manner. The data suggest that the fibrinolytic activity stimulated in whole human plasma in the presence of dextran sulfate and N-flufenamyl-β-alanine is dependent upon proteins of the contact activation system and also upon molecules immunologically related to urokinase.

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.

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

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.

Induction of Fibrinolysis by Polyanions in Human Plasma

1988 ◽  
Vol 60 (02) ◽  
pp. 324-327 ◽  
Author(s):  
Rainer J Klauser
Keyword(s):  
Human Plasma ◽  
Ribonucleic Acid ◽  
Fibrinolytic Activity ◽  
Dextran Sulfate ◽  
Factor Xii ◽  
Chromogenic Substrate ◽  
Pentosan Polysulfate ◽  
Hageman Factor ◽  
Plate Assay ◽  
Fibrin Plate

SummaryThe fibrinolytic potency of several polyanions was comparatively investigated. Fibrinolytic activity was measured in a whole plasma assay using H-D-Val-Leu-Lys-pNA (S-2251) as chromogenic substrate and by a fibrin plate assay using plasminogen rich fibrin plates.In the chromogenic substrate assay potent fibrinolytic polyanions comprised dextran sulfate, GAGPS, pentosan polysulfate, polyanethol sulfate, 1-carrageenan and i-carrageenan. Chon- droitin sulfates A, B, ‘C, keratan sulfate, ribonucleic acid, k-carrageenan and heparin were weakly fibrinolytic. Hyaluronic acid and lipopolysaccharide from E. coli were inactive.Similar results were obtained when fibrinolytic activity was measured by a fibrin plate assay. All polyanions except lipopolysaccharide produced lysis zones.Induction of fibrinolytic activity in human plasma was shown to be at least partially dependent on Hageman factor. In factor XII deficient plasma no fibrinolysis was induced by any of the polyanions when measured in the fibrin plate assay.In the. chromogenic substrate assay corn Hageman factor inhibitor (CHFI) inhibited the activation of S-2251 cleaving enzyme by GAGPS, pentosan poly sulfate, polyanethol sulfate, heparin, and ribonucleic acid near completely. The activation by dextran sulfate was inhibited by 45%.Heparin, pentosan polysulfate and GAGPS, three poly anions of therapeutic interest were separately compared. In both assays GAGPS proved the most potent activator, while pentosan poly sulfate exhibited 83% and 44% and heparin 32% and 14% of GAGPS fibrinolytic activity in the chromogenic substrate test and the fibrin plate assay, respectively.

scholarly journals Monoclonal antibody to human high-molecular-weight kininogen recognizes its prekallikrein binding site and inhibits its coagulant activity

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 695-702 ◽  
Author(s):  
SR Reddigari ◽  
AP Kaplan
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Binding Site ◽  
Human Plasma ◽  
Light Chain ◽  
Dextran Sulfate ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Normal Plasma ◽  
Coagulant Activity

We developed a mouse monoclonal antibody (MoAb 115–21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115–21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115–21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115–21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115– 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115–21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115–21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology.

scholarly journals Monoclonal antibody to human high-molecular-weight kininogen recognizes its prekallikrein binding site and inhibits its coagulant activity

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 695-702 ◽  
Author(s):  
SR Reddigari ◽  
AP Kaplan
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Binding Site ◽  
Human Plasma ◽  
Light Chain ◽  
Dextran Sulfate ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Normal Plasma ◽  
Coagulant Activity

Abstract We developed a mouse monoclonal antibody (MoAb 115–21) to human high- molecular-weight kininogen (HK) that recognizes its prekallikrein binding site (residues 565 through 595 of HK). The corresponding synthesized 31-amino acid peptide (peptide IV) was recently shown to retain native HK's prekallikrein binding property. The same peptide bound factor XI also, although less avidly. Our MoAb recognizes purified HK, peptide IV, and the light chain moiety of HK (where the peptide IV resides), as shown by enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. The apparent dissociation constant for the HK and MoAb 115–21 interaction was 2.2 nmol/L. It does not recognize low-molecular-weight kininogen (LK) with which HK shares its heavy chain moiety or any antigens in human plasma congenitally deficient in kininogens. The binding of MoAb 115–21 to purified light chain of HK was competitively inhibited by peptide IV. In addition, the antibody inhibits HK-dependent clotting activity of normal human plasma and dextran sulfate-mediated activation of prekallikrein in plasma and retards cleavage of HK in normal plasma after contact activation with dextran sulfate. Also, purified Fab fragments of MoAb 115–21 inhibited the HK-dependent coagulant activity and dextran sulfate-mediated prekallikrein activation in normal plasma. Since the kd for HK-MoAb 115– 21 interaction is ten times lower than that of HK-prekallikrein, our data suggest that binding of MoAb 115–21 to HK's peptide IV site increases the free prekallikrein concentration in plasma and thus results in the decreased efficiency of factor XIIa-mediated activation of prekallikrein. Decreased levels of kallikrein thus formed may be responsible for the inhibition of HK-dependent clotting activity and the decrease in rate and extent of HK cleavage in normal plasma on contact activation with dextran sulfate. MoAb 115–21 may thus prove very useful, especially with its high affinity for HK, in further delineation of the role of HK and prekallikrein in contact activation and kinin-related human pathology.

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.

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).

scholarly journals A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1053-1062
Author(s):  
D Veloso ◽  
LD Silver ◽  
S Hahn ◽  
RW Colman
Keyword(s):  
Heavy Chain ◽  
Sodium Dodecyl ◽  
Factor Xii ◽  
Contact Activation ◽  
Fab Fragments ◽  
Microtiter Plates ◽  
Normal Plasma ◽  
Reducing Conditions ◽  
Factor Xiia ◽  
Kallikrein Activity

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein- HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

HOMOZYGOUS FACTOR XII CONGENITAL DEFICIENCY: STUDY OF 10 NEW FAMILIES.

1987 ◽  
Author(s):  
G Castaman ◽  
F Rodeghiero ◽  
M Ruggeri
Keyword(s):  
Fibrinolytic Activity ◽  
Normal Values ◽  
Common Finding ◽  
Factor Xii ◽  
Normal Range ◽  
Congenital Deficiency ◽  
Fibrin Plate ◽  
Sporadic Cases ◽  
Laboratory Investigations ◽  
Normal Controls

Sporadic cases of thromboembolic events have been reported in patients with congenital factor XII deficiency and a relationship with a reduced intrinsic fibrinolysis has been suggested.We report here the results of clinical and laboratory investigations in 10 new families comprising 15 homozygotes (age 16-72) and 14 heterozygotes (age 18-65).In homozygotes, kaolin-activated-PTT was indefinitely prolonged and F XII activity and antigen were undetectable, whereas functional assays . of high molecular weight kininogen ahd kallikrein yielded normal values. Intrinsic fibrinolytic activity - assayed on fibrin plate by measuring lysis zones determined i. by euglobulin fraction, obtained in presence of dextran sulphate and flufenamate (Blood activator inventory test, Kluft 1979) - was reduced in all homozygous pts. to about 50% of normal (range 15-70%; normal range 80-120%); normal values were observed in all heterozygotes. Basal extrinsic fibrinolytic activity (measured after addition of Cl-inhibitor) was absent or minimal as in normal controls. None of our patients showed evidence of thrombotic diathesis.In conclusion, our study demonstrates that a reduced intrinsic fibrinolysis, as assayed by blood activator inventory test, is a common finding in F XII deficiency. The absence of thrombotic diathesis in our cases suggests that, this defect is probably devoid of any clinical significance.

scholarly journals A monoclonal anti-human plasma prekallikrein antibody that inhibits activation of prekallikrein by factor XIIa on a surface

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1053-1062 ◽  
Author(s):  
D Veloso ◽  
LD Silver ◽  
S Hahn ◽  
RW Colman
Keyword(s):  
Heavy Chain ◽  
Sodium Dodecyl ◽  
Factor Xii ◽  
Contact Activation ◽  
Fab Fragments ◽  
Microtiter Plates ◽  
Normal Plasma ◽  
Reducing Conditions ◽  
Factor Xiia ◽  
Kallikrein Activity

Abstract Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein- HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

Sign in / Sign up

Export Citation Format

Share Document