DETECTION AND QUANTITATION OF CLEAVED AND UNCLEAVED HIGH MOLECULAR WEIGHT KININOGEN IN PLASMA BY LIGAND BLOTTING WITH RADIOLABELED PLASMA PREKALLIKREIN OR FACTOR XI

1987 ◽  
Author(s):  
B Lämmle ◽  
B L Zuraw ◽  
M J Heeb ◽  
H P Schwarz ◽  
J G Curd ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Factor Xi ◽  
Sds Page ◽  
Normal Human

A method for the quantitative assay of native single chain and kallikrein cleaved two-chain high molecular weight kininogen (HMWK) in plasma has been developed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma is followed by electroblotting of the electropherogram to nitrocellulose membranes and detection of the inmobilized HMWK with its physiologic ligands, plasma prekallikrein or factor XI. Using 1251-prekallikrein or 125I-F.XI overlay nitrocellulose bound HMWK can be visualized by autoradiography.Using unreduced SDS-PAGE cleaved two-chain HMWK (Mr 107,000 and 95,000) is electrophoretically separated from uncleaved single chain HMWK (Mr 150,000). Counting the radioactivity of the nitrocellulose pieces corresponding to cleaved HMWK permits its quantitative measurement by comparison with standards consisting of decreasing amounts of fully dextran sulfate activated normal human plasma. Single chain HMWK is similarly assayed using reduced SDS-PAGE and unactivated normal human plasma standards.This technique is highly specific and sensitive to ˜ 50 ng of either cleaved or uncleaved HMWK. Varying concentrations of cleaved HMWK were found in plasmas from patients suffering from various systemic inflanmatory conditions. Higher levels of in vivo cleaved HMWK were observed during acute attacks of hereditary angioedema due to Cl-inhibitor deficiency.This technique may be useful for the assessment of the degree of in vitro or in vivo activation of the contact system of plasma.

Detection and Quantitation of Cleaved and Uncleaved High Molecular Weight Kininogen in Plasma by Ligand Blotting with Radiolabeled Plasma Prekallikrein or Factor XI

1988 ◽  
Vol 59 (02) ◽  
pp. 151-161 ◽  
Author(s):  
Bernhard Lämmle ◽  
Bruce L Zuraw ◽  
Mary Jo Heeb ◽  
Hans Peter Schwarz ◽  
Mauro Berrettini ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Sodium Dodecyl ◽  
Normal Human Plasma ◽  
Single Chain ◽  
Factor Xi ◽  
Sds Page ◽  
Normal Human

SummaryA method for the quantitative assay of native single chain and kallikrein cleaved two-chain high molecular weight (HMW)-kininogen in plasma is described. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma is followed by electrotransfer of the electropherogram to nitrocellulose membranes and detection of the blotted HMW-kininogen with its physiologic ligands, radiolabeled plasma prekallikrein or radiolabeled factor XI. Using unreduced SDS-PAGE cleaved two-chain HMW-kininogen (Mr ∼107,000 and 95,000), is elec-trophoretically separated from uncleaved single chain HMW-kininogen (Mr ∼150,000). Counting the radioactivity of the nitrocellulose pieces corresponding to cleaved HMW-kininogen permits its quantitative measurement by comparison with standards consisting of decreasing amounts of fully dextran sulfate activated normal human plasma. Single chain HMW-kininogen is similarly assayed using reduced SDS-PAGE and unactivated normal human plasma standards.This technique is highly specific and sensitive to about 50 ng of either cleaved or uncleaved HMW-kininogen. Varying amounts of cleaved HMW-kininogen were found in a small series of plasmas from patients suffering from various inflammatory conditions. Higher levels of in vivo cleaved HMW-kininogen were observed during acute attacks of hereditary angioedema due to Cl-inhibitor deficiency. This technique may be useful for the assessment of the degree of in vitro or in vivo 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.

The Levels of Factor XIIa Generated in Human Plasma on an Electronegative Surface Are Insensitive to Wide Variation in the Concentration of FXII, Prekallikrein, High Molecular Weight Kininogen or FXI

1999 ◽  
Vol 82 (09) ◽  
pp. 1033-1040 ◽  
Author(s):  
K. A. Mitropoulos
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Bulk Phase ◽  
Factor Xii ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Wide Range ◽  
Factor Xiia ◽  
Normal Human

SummaryThe contribution of the various components of the contact system in the generation of factor XIIa (FXIIa) and of kallikrein (KRN) on an electronegative surface and the release of the generated enzymes to the bulk phase was examined in mixtures of normal human plasma and plasmas congenitally deficient in these components. The incubation of normal human plasma in the presence of sulphatide vesicles (40 μM) resulted in a fast generation of amidolytic activities due to FXIIa and to KRN followed by slower first-order inactivation rates of FXIIa (k’FXIIa) and of KRN (k’KRN) due to the presence of esterase inhibitors. Variation of the levels of factor XII (FXII), over a wide range, showed little effect on levels of FXIIa and of KRN but no activities were detected in 100% FXII-deficient plasma. The variation of prekallikrein (PKRN) concentration showed little effect on the generation of FXIIa but the generation of KRN declined linearly with the decrease in the level of PKRN. No activities were detected on treatment of PKRN-deficient plasma. The variation in the concentration of high molecular weight kininogen (HK) showed effects on FXIIa and KRN that were qualitatively similar to those seen on variation of PKRN but 100% HK-deficient plasma generated considerable activities of both FXIIa and KRN. The variation in the concentration of factor XI (FXI) showed no effect on the generation of FXIIa, whereas KRN levels increased linearly with the contribution of FXI-deficient in normal plasma. The present results suggest that the contiguous binding of FXIIa, FXII, PKRN-HK and FXI-HK onto the electronegative surface induces a rapid generation of FXIIa and KRN. The bound PKRN-HK complex prevents the release of generated FXIIa and therefore further binding and activation of FXII from the bulk phase. Consequently, the turnover of FXII is independent of its levels in the bulk phase and is rather related to the concentration of contact surface. The generated KRN is also protected by HK. However, since the enzyme responsible for the activation of PKRN-HK is FXIIa, the levels of generated KRN are positively related to the concentration of substrate.

scholarly journals High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 500-507 ◽  
Author(s):  
RN Puri ◽  
F Zhou ◽  
CJ Hu ◽  
RF Colman ◽  
RW Colman
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Plasma Concentration ◽  
Human Plasma ◽  
Shape Change ◽  
Dependent Manner ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Normal Human ◽  
Dose Dependent

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)- induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin- induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma- thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma- thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

Bacterial Expression of Biologically Active High Molecular Weight Kininogen Light Chain

1992 ◽  
Vol 67 (04) ◽  
pp. 428-433 ◽  
Author(s):  
Satya P Kunapuli ◽  
Raul A DeLa Cadena ◽  
Robert W Colman
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Light Chain ◽  
Specific Activity ◽  
Bacterial Expression ◽  
Contact System ◽  
Biologically Active ◽  
High Molecular Weight Kininogen ◽  
Single Chain

SummaryHuman high molecular weight kininogen (HK), a single chain plasma glycoprotein, serves as a cofactor in the contact system of blood coagulation. After cleavage by human plasma kallikrein, the nonapeptide bradykinin is released. The HK light chain (LC) contains coagulant activity, which requires both the ability to bind the contact system zymogens, prekallikrein and factor XI, and the ability to interact with negatively charged surfaces. Since bacterial expression might not be successful if carbohydrate was required for activity, we evaluated that possibility by incubating plasma HK with endoglycosydase F. Although the procedure removed detectable N-linked carbohydrate, no change in specific activity occurred. We then developed a bacterial expression system to produce recombinant HK LC. The cDNA coding for the HK LC was prepared by polymerase chain reaction (PCR), digested with restriction enzymes EcoRI and PstI, and introduced into the bacterial expression vector pKK223-3. E. coli harboring this recombinant plasmid (pSKl) expressed HK LC upon induction with isopropylthio-galactoside (IPTG). The recombinant protein (27 kDa), when transferred onto a PVDF membrane, was recognized by monospecific polyclonal anti-HK LC-antibodies. The recombinant HK LC was purified by heparin agarose affinity chromatography to homogeneity and found to have a specific activity of 28 coagulant units per mg protein, similar to the specific activity of the LC derived by proteolytic digestion of human plasma HK. We conclude: 1) The HK LC synthesized in bacteria is biologically active, and 2) the 40% carbohydrate content of the HK LC is not required for its cofactor activity.

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