Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

Rabbit Plasma, unlike Its Human Counterpart, Contains no Complex between Protein S and C4b-Binding Protein

1994 ◽  
Vol 71 (04) ◽  
pp. 446-451 ◽  
Author(s):  
Xuhua He ◽  
Björn Dahlbäck
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Complex Formation ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Western Blotting ◽  
Vitamin K ◽  
Protein S ◽  
Rabbit Plasma ◽  
Free Protein

SummaryIn human plasma, the anticoagulant vitamin K-dependent protein S exists in two molecular forms, as free protein and complexed to C4b- binding protein (C4BP), a complement regulatory protein. It has been suggested that rabbit plasma also contains two forms of protein S and that the interaction between protein S and C4BP m rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-lerminal part of protein S. In this report, we provide itsulls which challenge the conclusion that rabbit plasma contains the complexed form of protein S. The two forms of protein S in human plasma were separated by gel filtration chromatography on Sephacryl S-300 and the presence of protein S in the various fractions analyzed by Western blotting using a monoclonal antibody (HPS 21) directed against the γ-carboxyglutamic acid rich module of human protein S. This antibody, which was found to cross-react with rabbit protein S on Western blotting, was used in affinity purification of protein S from rabbit plasma as well as of recombinant rabbit protein S. HPS 21 specifically recognized protein S in rabbit plasma and did not cross-react with the other vitamin K-depeudenl plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gelfiltration chromatography followed by Western blotting of the fractions with monoclonal antibody HPS 21. Protein S was found only in fractions eluting at a position corresponding to that of free protein S. A radiolabelled trace amount of recombinant rabbit protein S added to rabbit plasma chromatographed as free protein S and no high molecular weight form corresponding to a C4BP-protein S complex was detected. Rabbit protein S had the capacity to bind human C4BP and the addition of human C4BP to rabbit plasma changed the elution profile, of rabbit plasma protein S. After the addition of human C4BP, rabbit plasma protein S quantitatively eluted as a high molecular weight complex, suggesting that all the protein S in rabbit plasma was bound to human C4BP. The anticoagulant activity of human protein S is modulated by the complex formation with C4BP. Our results demonstrate that this function of C4BP and the protein S-C4BP complex formation has not been conserved throughout the evolution even though protein S has a preserved C4BP binding site.

scholarly journals Monoclonal antibodies to discrete regions in alpha 2-plasmin inhibitor

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 446-453 ◽  
Author(s):  
J Mimuro ◽  
Y Koike ◽  
Y Sumi ◽  
N Aoki
Keyword(s):  
Monoclonal Antibodies ◽  
Complex Formation ◽  
Binding Site ◽  
Factor Xiii ◽  
Cross Linking ◽  
Reactive Site ◽  
The Third ◽  
N Terminus ◽  
Mechanism Of Interaction ◽  
Plasmin Inhibitor

Abstract Three monoclonal antibodies to alpha 2-plasmin inhibitor (alpha 2PI) were characterized. The first, JTPI-1, was directed against the reative site of alpha 2PI and inhibited antiplasmin activity by interfering with the formation of alpha 2PI-plasmin complexes. The avidity of JTPI- 1 to the preformed alpha 2PI-plasmin complex was markedly lower than that to free alpha 2PI, which made this antibody useful for measuring the free alpha 2PI in plasma. The second, JTPI-2, recognized an epitope in the C-terminal fragment of alpha 2PI (11,000 daltons [11 K]) that was cleaved from alpha 2PI by plasmin upon complex formation but remained noncovalently attached to the complex. However, binding of JTPI-2 to alpha 2PI was not inhibited by the C-terminal 26-residue peptide containing the plasminogen-binding site and had no effect on the function of alpha 2PI. These data suggested that JTPI-2 recognized an epitope between the C-terminal 26-residue peptide and the reactive site. The third, JTPI-3, bound the alpha 2PI-plasmin complex (150 K) as well as alpha 2PI. Binding was inhibited by the N-terminal 12-residue peptide of alpha 2PI, but factor XIII-catalyzed cross-linking of alpha 2PI to fibrin was not inhibited by JTPI-3. These results suggested that the antibody recognized an epitope near the N terminus. These three monoclonal antibodies were useful for analyzing the mechanism of interaction between alpha 2PI and plasmin.

Soluble High-Molecular-Weight E Fragments in the Plasmin-Induced Degradation Products of Cross-Linked Human Fibrin

1975 ◽  
Vol 49 (2) ◽  
pp. 149-156 ◽  
Author(s):  
P. J. Gaffney ◽  
D. A. Lane ◽  
M. Brasher
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
D Dimer

1. The factor XIII-mediated cross-linked α chains in fibrin have no effect on the nature of the fragments released during the solubilization of fibrin by plasmin. 2. Besides the known D dimer and E fragments solubilized during the lysis of cross-linked fibrin, other fragments have been observed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which have a molecular weight of about 135 000. After prolonged plasmin digestion, these fragments (U fragments) were no longer evident on the gels and the high-molecular-weight E antigen was absent. It is assumed that the E antigen was associated with the U fragments. These fragments also cross-reacted with an anti-D serum. 3. The U fragments have been tentatively presumed to be a factor XIII-mediated cross-linked D–E complex since they degrade only after prolonged degradation with plasmin. Whereas it is known that the fibrin D dimer fragment contains the cross-linked γ chain residues of the originating fibrin, the presumed covalent cross-linking of the D–E fragments has not been proved. 4. The presence of these high-molecular-weight fragments, containing the E antigen, in cross-linked human fibrin digests should be taken into account in the development of D dimer assays to monitor fibrin lysis in vivo.

scholarly journals Monoclonal antibodies to discrete regions in alpha 2-plasmin inhibitor

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 446-453
Author(s):  
J Mimuro ◽  
Y Koike ◽  
Y Sumi ◽  
N Aoki
Keyword(s):  
Monoclonal Antibodies ◽  
Complex Formation ◽  
Binding Site ◽  
Factor Xiii ◽  
Cross Linking ◽  
Reactive Site ◽  
The Third ◽  
N Terminus ◽  
Mechanism Of Interaction ◽  
Plasmin Inhibitor

Three monoclonal antibodies to alpha 2-plasmin inhibitor (alpha 2PI) were characterized. The first, JTPI-1, was directed against the reative site of alpha 2PI and inhibited antiplasmin activity by interfering with the formation of alpha 2PI-plasmin complexes. The avidity of JTPI- 1 to the preformed alpha 2PI-plasmin complex was markedly lower than that to free alpha 2PI, which made this antibody useful for measuring the free alpha 2PI in plasma. The second, JTPI-2, recognized an epitope in the C-terminal fragment of alpha 2PI (11,000 daltons [11 K]) that was cleaved from alpha 2PI by plasmin upon complex formation but remained noncovalently attached to the complex. However, binding of JTPI-2 to alpha 2PI was not inhibited by the C-terminal 26-residue peptide containing the plasminogen-binding site and had no effect on the function of alpha 2PI. These data suggested that JTPI-2 recognized an epitope between the C-terminal 26-residue peptide and the reactive site. The third, JTPI-3, bound the alpha 2PI-plasmin complex (150 K) as well as alpha 2PI. Binding was inhibited by the N-terminal 12-residue peptide of alpha 2PI, but factor XIII-catalyzed cross-linking of alpha 2PI to fibrin was not inhibited by JTPI-3. These results suggested that the antibody recognized an epitope near the N terminus. These three monoclonal antibodies were useful for analyzing the mechanism of interaction between alpha 2PI and plasmin.

RECOMBINANT AND PLASMA FVIII SHARE EPITOPES RECOGNIZED BY A PANEL OF MONOCLONAL AND POLYCLONAL ANTIBODIES

1987 ◽  
Author(s):  
N Pancham ◽  
M Dumas ◽  
M S Madant ◽  
P C Esmon ◽  
V Voehrinqer
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Functional Characteristics ◽  
Activity Inhibition ◽  
One Stage ◽  
Recombinant Fviii

A panel of neutralizing and nonneutralizing monoclonal and polyclonal antibodies raised against human plasma FVIII and human recombinant FVIII was employed in Western blotting, activity inhibition and two-site ELISA studies to investigate structural similarities between recombinant FVIII (r-FVIII) and plasma FVIII (p-FVIII). The monoclonal antibodies were obtained either commercially or produced in-house and react with either the 90kd, 80kd or B-region polypeptides. Some antibodies were raised to FVIII synthetic peptides. Rabbit polyclonal antibodies were obtained from either r-FVIII or p-FVIII immunizations. Recombinant FVIII and p-FVIII were compared either as intact molecules or following thrombin digestion by immunoblots after SDS gradient PAGE. Combinations of antibodies were used in two-site ELISA. Activity inhibition was evaluated by one-stage clotting or in FXa generation assays. Recombinant FVIII and p-FVIII responded similarly to these antibodies in all of the immunotechniques used. Collectively, these results indicate that using this approach r-FVIII and p-FVIII display similar structural and functional characteristics.

The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 988-995 ◽  
Author(s):  
Robert A. S. Ariëns ◽  
Helen Philippou ◽  
Chandrasekaran Nagaswami ◽  
John W. Weisel ◽  
David A. Lane ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Factor Xiii ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Similar Rate ◽  
Cross Linking ◽  
Fibrinopeptide A ◽  
Thrombin Cleavage

Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (kcat/Km) of thrombin was 0.5 for FXIII 34Leu and 0.2 (μmol/L)−1× sec−1 for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (μmol/L)−1 × sec−1, respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of γ- and -chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.

Fibrin Detected in Plasma of Patients with Disseminated intravascuiar Coagulation by Fibrin-specific Antibodies Consists Primarily of High Molecular Weight Factor XIIIa-crosslinked and Plasmin-modified Complexes Partially Containing Fibrinopeptide A

1997 ◽  
Vol 78 (03) ◽  
pp. 1069-1078 ◽  
Author(s):  
Susanne A Pfitzner ◽  
Carl-Erik Dempfle ◽  
Michio Matsuda ◽  
Dieter L Heene
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Factor Xiiia ◽  
Fibrinopeptide A ◽  
Weight Factor ◽  
Fibrin Monomer ◽  
N Terminus ◽  
Α Chain ◽  
D Dimer

SummaryPooled plasma from 40 patients with severe disseminated intravascuiar coagulation (DIC) secondary to septic conditions was subjected to gel permeation chromatography on Sephacryl S-500 HR after sample pretreatment with KSCN for dissociation of non-covalent fibrin complexes. Fibrin antigen in eluates was detected by an array of ELISA tests, using two monoclonal antibodies against fibrin degradation product D-dimer, a monoclonal antibody against an epitope generated by plasmin cleavage of the D-domain, and an antibody against the neo-N- terminus of the α-chain of fibrin exposed by cleavage of fibrinopeptide A. Tag antibodies were a polyclonal antibody against the fibrinogen/ fibrin D-domain, a POD-conjugated version of the monoclonal antibody against fibrin α-chain neo-N-terminus, and a polyclonal antibody against fibrinopeptide A. Most fibrin-related material present in the pooled DIC plasma was of higher molecular mass than fibrinogen. Fibrin polymers were reactive with antibodies against D-dimer, plasmin cleaved D-domain, and fibrin α-chain neo-N-terminus. Part of the polymers reacted with antibodies against fibrinopeptide A, indicating presence of fibrinogen or desA-fibrin monomer within the covalently linked complex. In conclusion, the primary analytes detected by monoclonal antibodies for D-dimer, plasmin-specific epitopes of fibrin degradation products, as well as sites exposed by fibrinopeptide cleavage in plasma from patients with disseminated intravascuiar coagulation are high molecular weight factor XIIIa-crosslinked fibrin complexes, containing plasmin-cleaved D-domains, intact fibrin monomer units, and fibrinogen or desA-fibrin monomer.

Comparison of the Cross-Linking Patterns of Lamprey Fibrinogen and Fibrin by the Action of the Intrinsic Lamprey Factor XIII and Human Factor XIII during the Process of Blood Coagulation

1977 ◽  
Vol 38 (02) ◽  
pp. 0429-0437 ◽  
Author(s):  
Patricia A. Murtaugh
Keyword(s):  
Molecular Weight ◽  
Blood Coagulation ◽  
High Molecular Weight ◽  
Factor Xiii ◽  
Human Factor ◽  
Cross Linking ◽  
High Molecular Weight Polymer ◽  
Cross Links ◽  
Α Chain ◽  
A Chain

SummaryIntrinsic lamprey factor XIII cross-links the γ-chain of lamprey fibrin (50,000 daltons) to the γ-dimer (100,000 daltons). The a-chain (110,000 daltons) is cross-linked very slowly to a-dimer (210,000 daltons) and a-trimer (330,000 daltons). In contrast, human factor XIII, when added in combination with intrinsic lamprey factor XIII, cross-finks the a-chain of lamprey fibrin to a high molecular weight polymer, and any remaining γ-chain is also cross-linked to a polymer. However, the γ-chain that has previously cross-linked to the γ-dimer by the intrinsic lamprey factor XIII remains as a γ-dimer. Factor XIII-free lamprey fibrin cross-links all its subunits (α, β, γ) to high molecular weight polymers when human factor XIII is added. In contrast to human and bovine fibrin where α-chain cross-linking in the process of blood coagulation commences when all of the γ-chain has cross-linked, the lamprey α-chain will begin to cross-link when approximately half of the γ-chain has cross-linked to the γ-dimer.

The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 988-995 ◽  
Author(s):  
Robert A. S. Ariëns ◽  
Helen Philippou ◽  
Chandrasekaran Nagaswami ◽  
John W. Weisel ◽  
David A. Lane ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Factor Xiii ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Similar Rate ◽  
Cross Linking ◽  
Fibrinopeptide A ◽  
Thrombin Cleavage

Abstract Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (kcat/Km) of thrombin was 0.5 for FXIII 34Leu and 0.2 (μmol/L)−1× sec−1 for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (μmol/L)−1 × sec−1, respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of γ- and -chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.

Increased levels of a lower molecular weight form of secretory component in cervical scrapes from women with cervical intraepithelial neoplasia

1993 ◽  
Vol 3 (4) ◽  
pp. 253-258 ◽  
Author(s):  
J. C. Longfield ◽  
L. M.H. Jones ◽  
S. Thompson ◽  
J. M. Monaghan ◽  
G. A. Turner
Keyword(s):  
Molecular Weight ◽  
Cervical Intraepithelial Neoplasia ◽  
Western Blotting ◽  
Quantitative Method ◽  
Protein Composition ◽  
Intraepithelial Neoplasia ◽  
Secretory Component ◽  
Lower Molecular Weight ◽  
2D Electrophoresis ◽  
Molecular Weight Form

The protein composition of ‘negative’ and ‘positive’ cervical scrapes has been compared using electrophoresis to determine whether differences were present that could be used to pre-select specimens with negative cytology. Only one minor difference in the 75–80 kDa region of the silver-stained patterns was detected in extracts from two well-matched groups. Further studies using 2D-electrophoresis and Western blotting identified the major components in this region as two forms of secretory component (SC) and transferrin. Subsequent blotting of 13 extracts from patients with negative smears and 17 extracts from patients with positive smears indicated very significant differences in the expression of the two forms of SC (P< 0.0002), the ‘negatives’ having more of the higherMr form and the ‘positives’ having more of the lowerMr form. Measuring SC could prove useful for pre-screening cervical scrapes, but more investigations are required to establish the nature of the change and a quantitative method for its detection.

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