INVOLVEMENT OF FACTOR XII (F XII) AND PREKALLIKREIN (PKK) IN THE ACTIVATION OF UROKINASE (UK)-RELATED PROTEINS IN HUMAN PLASMA.

1987 ◽  
Author(s):  
D J Binnema ◽  
G Dooijewaard
Keyword(s):  
Human Plasma ◽  
Specific Activity ◽  
Antigenic Determinants ◽  
Gel Chromatography ◽  
Type I ◽  
Factor Xii ◽  
Single Chain ◽  
Sds Page ◽  
The Uk ◽  
Related Proteins

Recently it has been shown that in human plasma two types of UK-related proteins occur: Type I, plasma UK, with UK-related antigenic determinants directly accessible to anti-UK antibodies and Type II with UK-related antigenic determinants which become accessible only after SDS treatment and separation of polypeptides on PAGE. In this study we compared the molecular and enzymic properties of the two types in: 1. plasma activated by dextran sulphate (DXS) euglobulin precipitation, 2. plasma that was not activated and 3. plasma deficient in F XII, depleted in PKK and subsequently activated by DXS. ACA 34 gel chromatography, SDS PAGE, fibrin underlay zymography and immunoblotting were used. Results:Conclusions: 1. The UK-related subunits of T1 and TII are active when cleaved, but relatively inactive in the single-chain form. 2. The presence of F XII and PKK is indispensable for activation of TII, but not for that of TI; TII contributes to the F Xll-de-pendent plasminogen activator activity reported earlier, TI to the F Xll-independent part. 3. Activation of TI by DXS with no F XII and PKK present impairs the formation of the 150,000 form. 4. The specific activity of TII is rather low, but its concentration in plasma (not shown) is at least ten times that of TI.

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 Protease activity in single-chain prekallikrein

Blood ◽  
2020 ◽  
Vol 135 (8) ◽  
pp. 558-567 ◽  
Author(s):  
Ivan Ivanov ◽  
Ingrid M. Verhamme ◽  
Mao-fu Sun ◽  
Bassem Mohammed ◽  
Qiufang Cheng ◽  
...  
Keyword(s):  
Specific Activity ◽  
Catalytic Efficiency ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Single Chain ◽  
Similar Activity ◽  
Artificial Surfaces ◽  
Protease Precursor ◽  
Enzyme Cascades

Abstract Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

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.

The Contact-System Dependent Plasminogen Activator from Human Plasma: Identification and Characterization

1990 ◽  
Vol 64 (03) ◽  
pp. 390-397 ◽  
Author(s):  
D J Binnema ◽  
G Dooijewaard ◽  
J J L van Iersel ◽  
P N C Turion ◽  
C Kluft
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Physiological Role ◽  
Contact System ◽  
Tissue Type ◽  
Antigenic Determinants ◽  
Dextran Sulphate ◽  
Single Chain ◽  
Native Form ◽  
Type Plasminogen Activator

SummaryApart from tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), a third PA appears to occur in human plasma. Its activity is initiated when appropriate triggers of the contact system are added, and the activation depends on the presence of factor III and prekallikrein in plasma. The activity of this, so-called, contact-system dependent PA accounts for 30% of the PA activity in the dextran sulphate euglobulin fraction of plasma and was shown not to be an intrinsic property of one of the contact-system components, nor could it be inhibited by inhibitory antibodies against t-PA or u-PA. We have succeeded in identifying this third PA in dextran sulphate euglobulin fractions of human plasma. Its smallest unit (SDSPAGE) is an inactive 110 kDa single-chain polypeptide which upon activation of the contact system is converted to a cleaved, disulphide-bridged molecule with PA activity. The native form, presumably, is an oligomer, since the apparent M. on gelchromatography is 600,000. The IEP is 4.8, much lower than that of t-PA and u-PA. Although the active 110 kDa polypeptide cannot be inhibited by anti-u-PA, it yet comprises a 37 kDa piece with some u-PA related antigenic determinants. However, these determinants are in a latent or cryptic form, only detectable after denaturation by SDS. The 110 kDa polypeptide is evidently not a dimer of 55 kDa u-PA or a complex of u-PA with an inhibitor. It is probably a PA derived from a gene quite distinct from that of t-PA or u-PA, but sharing some homology with u-PA. The physiological role of this contact-system dependent PA remains to be established

An Analysis of the Activators of Single-Chain Urokinase-Type Plasminogen Activator (scu-PA) in the Dextran Sulphate Euglobulin Fraction of Normal Plasma and of Plasmas Deficient in Factor XII and Prekallikrein

1991 ◽  
Vol 65 (02) ◽  
pp. 144-148 ◽  
Author(s):  
D J Binnema ◽  
G Dooijewaard ◽  
P N C Turion
Keyword(s):  
Plasminogen Activator ◽  
Gel Chromatography ◽  
Factor Xii ◽  
Dextran Sulphate ◽  
Contact Activation ◽  
Single Chain ◽  
Activity Peak ◽  
Normal Plasma ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryAn analysis was made of the various possible activators of single-chain urokinase-type plasminogen activator (scu-PA) in the dextran sulphate euglobulin fraction (DEF) of human plasma. scu-PA activators were detected in an assay system in which the substrate scu-PA, in physiological concentration (50 pM), was immuno-immobilized. After activation of the immobilized scu-PA for a certain period of time the activity of the generated amount of immuno-immobilized two-chain u-PA was determined with plasminogen and the chromogenic substrate S-2251. The scu-PA activator activity (scuPA-AA) in the DEF of plasmas deficient in factor XII or prekallikrein was about half of that in the DEF of normal plasma. Separation of scuPA-AA in the DEF by gel chromatography showed to major peaks, one eluting with an apparent Mr of 500,000 and the other around Mr 100,000. The former peak, which coincided with the activity peak of the kallikrein-kininogen complex, was absent in the DEF of plasma depleted of prekallikrein and therefore was identified as kalli-krein. The latter peak was still present in the depleted plasma and most likely represents plasmin, because its scuPA-AA coincided with the activity peak of plasmin and could be fully inhibited by antibodies raised against human plasminogen. It is concluded that plasmin and the contact-activation factor kallikrein each contribute for about 50% to the scuPA-AA in the DEF. Compared on a molar basis, however, plasmin was found to be almost 1,000 times more effective than kallikrein, and we conclude, therefore, that in vivo plasmin is the primary activator of scu-PA and the role of the contact system is of secondary importance.

Studies on the Biochemistry of Urokinase

1977 ◽  
Vol 38 (04) ◽  
pp. 0801-0808 ◽  
Author(s):  
Eng Bee Ong ◽  
Mercedes E. Soberano ◽  
Alan J. Johnson ◽  
Guenther Schoellmann
Keyword(s):  
Affinity Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Blue Staining ◽  
Single Chain ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Sds Page ◽  
Excess Substrate

SummaryDirect evidence for an active center histidine residue in urokinase (UK) was obtained with use of newly synthesized peptide chloroketones Ac-Gly-Lys-CH2C1 and Nle-Gly-Lys-CH2C1. Stoichiometric inactivation by DFP provided further evidence that UK is a serine protease. Essential histidine and serine residues were both located in the heavy chain of the 47,0 M. W.UK. The high M.W. form can be converted (catalytically) to the low M. W. form.9 partially purified human urinary UK preparations (5 with predominantly high M. W. UK), varying in purity and proportion of high and low M. W. forms, were found to be heterogeneous by a number of acrylamide electrophoretic procedures. 7 preparations had strikingly similar molar activities at excess substrate, except for the lower values found in 2 predominantly high M. W. UK preparations from the same supplier. 2 high M. W. UK preparations from another supplier showed a definite increase in activity when assayed at low plasminogen concentration, but this effect was abolished after gel filtration (Sephadex G-25), by further purification with affinity chromatography, or when assayed with excess plasminogen.The high and low M. W. forms of UK (47,000 and 33,400 M. W.), isolated and purified by Sepharose-EACA-agmatine affinity chromatography were shown to be homogeneous by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis (PAGE) and by 14C-DFP and 14C-NPGB incorporation before SDS-PAGE. Comparative properties of the high M. W. vs low M. W. forms were as follows: specific activity (104,000 IU/mg vs 226,000) ; 2 chains (33,100 and 18,600 M.W.) linked by disulfide(s) vs a single chain; pi 8.60 (major subform) and pi 8.90 (minor subform) vs pi 8.35, 8.60, 8.70 (major subform) and pi 8.05 (minor subform); and second order kinetics for DFP inactivation (400 vs 770 M−1 min−1). The molar activities were similar (9.6 × 109 and 10.2 × 109IUm/mole) for each form.

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.

scholarly journals Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 455-462 ◽  
Author(s):  
M Berrettini ◽  
B Lammle ◽  
T White ◽  
MJ Heeb ◽  
HP Schwarz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Heavy Chain ◽  
Light Chain ◽  
Plasma Samples ◽  
Single Chain ◽  
Sds Page ◽  
Immunoblotting Technique

Abstract Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

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.

Purification and Characterization of Single-Chain Urokinase-Type Plasminogen Activator (Pro-Urokinase) from Human A431 Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 219-224 ◽  
Author(s):  
Angelo Corti ◽  
Maria Luisa Nolli ◽  
Adolfo Soffientini ◽  
Giovanni Cassani
Keyword(s):  
Plasminogen Activator ◽  
Specific Activity ◽  
Active Form ◽  
Human Kidney ◽  
Single Chain ◽  
A431 Cells ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Sds Page ◽  
Aminoacid Sequence

SummaryA single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified ˜18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 jig of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pi 9.05 and pi 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I.U./mg.

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