On the Plasmin Digestion of Factor XIII

1975 ◽  
Author(s):  
K. Mikami ◽  
T. Mikami ◽  
H. Suzuki ◽  
M. Fujimaki ◽  
K. Fukutake
Keyword(s):  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Platelet Factor ◽  
Subunit A ◽  
Plasma Factor ◽  
Congenital Afibrinogenemia

The plasmin digestion of factor XIII in the plasma with congenital afibrinogenemia has been reported by Suzuki et al. in 1967. The present investigation using polyacrylamide gel electrophoresis and crossimmunoelectrophoresis with anti-factor XIII (A & S) serum demonstrates that the process of plasmin digestion of factor XIII can be divided into three steps ; the subunit A of factor XIII such as placental or platelet factor XIII is ready to get plasmin digestion following the decrease of amount and activity of subunit A, and the subunit S in the A1 S complex like plasma factor XIII is more readily affectable on the plasmin digestion than the subunit A of the complex, and the A2 S complex coexisting with fibrinogen or plasma protein is fairly stable on exposure to plasmin.

scholarly journals Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with α2-macroglobulin

1980 ◽  
Vol 191 (3) ◽  
pp. 799-809 ◽  
Author(s):  
R G Sutcliffe ◽  
B M Kukulska-Langlands ◽  
J R Coggins ◽  
J B Hunter ◽  
C H Gore
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Plasma Protein ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Carbohydrate Content ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Tryptic Fragment

Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000–820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A.

scholarly journals In vivo radiolabeling of platelet proteins: a new method with identification of platelet factor XIII

Blood ◽  
1980 ◽  
Vol 55 (4) ◽  
pp. 559-563 ◽  
Author(s):  
DM Rider ◽  
RP McDonagh ◽  
J McDonagh
Keyword(s):  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Platelet Factor ◽  
Intravenous Injections ◽  
Molecular Weights ◽  
The Third

Abstract A method for radiolabeling platelets in vivo was developed in which 3H- arginine was injected into the bone marrow of normal dogs. On the third day after injection, a maximum of 6%--7% of the radioactivity had been incorporated into the total platelet mass. This method of isotope administration resulted in a 50--60-fold increase in maximum uptake of radiolabel by platelets, as compared to values obtained by others using intravenous injections of various radioactive compounds. Tritium- labeled platelets were harvested from the animals and then were washed to remove unbound 3H-arginine. On polyacrylamide gel electrophoresis 7 labeled protein bands, with molecular weights ranging from 29,000to 132,000, were obtained from the platelet-soluble fraction. One 3H- containing protein with a molecular weight of 81,000 was identified immunologically and enzymatically as platelet factor XIII.

scholarly journals Regulation of plasma factor XIII binding to fibrin in vitro

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1028-1034 ◽  
Author(s):  
CS Greenberg ◽  
JV Dobson ◽  
CC Miraglia
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protamine Sulfate ◽  
Factor Xiiia ◽  
Plasma Sodium ◽  
Platelet Factor ◽  
Plasma Factor

Abstract The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2- subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.

Platelet Factor XIII Becomes Active without the Release of Activation Peptide during Platelet Activation

1993 ◽  
Vol 69 (03) ◽  
pp. 282-285 ◽  
Author(s):  
László Muszbek ◽  
János Polgár ◽  
Zoltán Boda
Keyword(s):  
Platelet Activation ◽  
Factor Xiii ◽  
Platelet Factor ◽  
Subunit A ◽  
Cellular Factor ◽  
B Subunit ◽  
Exact Function ◽  
Plasma Factor ◽  
A Subunit ◽  
Activation Peptide

SummaryThe potentially active A subunit of factor XIII of blood coagulation has also been detected in platelets and monocytes/macrophages though the exact function of this cellular protransglutaminase has not yet been elucidated. In physiological conditions the first step in the activation of plasma factor XIII is the removal of an activation peptide from the N-terminal end of subunit A by thrombin. The A subunit then, in the presence of Ca2+, dissociates from the inhibitory B subunit and assumes an active conformation. Cellular factor XIII, which lacks B subunit, can be proteolytically activated in vitro by thrombin and the intracellular Ca2+ sensitive protease, calpain, in the same way as plasma factor XIII subunit A, and calpain has been suggested as the intracellular protease involved in the activation of cellular factor XIII in platelets. In the present experiments it was shown by SDS PAGE that during long-term stimulation of platelets with thrombin nondisulfide-crosslinked high M r protein polymers not penetrating the concentrating gel were formed. The lack of these polymers in thrombin-stimulated factor XIII deficient platelets clearly indicated that their formation in normal platelets was due to factor XIII that became active during platelet activation. However, no release of the activation peptide could be detected by Western blotting during this process. Similarly, no proteolytic cleavage of factor XIII was detectable when platelets were stimulated by Ca2+ ionophore through this stimulus activated calpain as it was clearly demonstrated by the breakdown of major intracellular calpain substrates. The results indicate: 1) during thrombin induced platelet activation factor XIII becomes active and crosslinks platelet protein, 2) platelet factor XIII is not an intracellular substrate of calpain, 3) cellular factor XIII could be activated without the proteolytic removal of activation peptide. It is presumed that the nonproteolytic pathway for the activation of cellular factor XIII, we reported most recently, might have physiological implications under such conditions.

scholarly journals Regulation of plasma factor XIII binding to fibrin in vitro

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1028-1034 ◽  
Author(s):  
CS Greenberg ◽  
JV Dobson ◽  
CC Miraglia
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protamine Sulfate ◽  
Factor Xiiia ◽  
Plasma Sodium ◽  
Platelet Factor ◽  
Plasma Factor

The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2- subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.

Reactivity of Fibrinogen Crosslinking Sites in the Absence of Thrombin

1976 ◽  
Vol 35 (03) ◽  
pp. 620-627 ◽  
Author(s):  
Thomas Seelich ◽  
Miha Furlan ◽  
Eugen A. Beck
Keyword(s):  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Conformational State ◽  
Protamine Sulfate ◽  
Polyacrylamide Gel ◽  
Physiological Conditions

SummaryThe reactivity of fibrinogen crosslinking sites with thrombin-free, preactivated factor XIII (F. Xllla) was investigated under different conditions such as increased ionic strength, presence of urea, protamine sulfate (PS) or of varying concentrations of monodansylcadaverine (MDC). Crosslinking and incorporation of MDC into fibrinogen or fibrin γ- and α-chains were evaluated by SDS-Polyacrylamide gel electrophoresis.According to our results, rates of crosslinking of, and of MDC incorporation into, both γ-and α-chains of fibrinogen were low under physiological conditions; they were not significantly influenced by the presence of either 1.0 M NaCl or 1.0 M urea. In contrast, 0.01 % PS precipitated fibrinogen, and, simultaneously, significantly increased the rates of crosslinking and of MDC incorporation into both γ- and α-chains. MDC, at concentrations above approximately 6 mM, also precipitated fibrinogen, and, up to a concentration of about 9 mM, markedly enhanced the reactivity of acceptor crosslinking sites.Our results suggest that solubility of fibrinogen and the conformational arrangement of its subunit chains are closely interdependent; the reactivity of crosslinking sites with F. Xllla seems to be a function of this conformational state.

Reactivity of Fibrinogen Acceptor Crosslinking Sites in the Absence of Thrombin

1975 ◽  
Author(s):  
T. Seelich ◽  
M. Furlan ◽  
E. A. Beck
Keyword(s):  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
High Ionic Strength ◽  
Protamine Sulfate ◽  
Polyacrylamide Gel

The interaction of thrombin-free, solid-phase preactivated factor XIII with fibrinogen was followed by SDS-polyacrylamide gel electrophoresis. Effects of increased ionic strength, protamine sulfate, or urea were evaluated both in the presence or absence of monodansyleadaverine.Our results suggest that high ionic strength in the absence of thrombin alters fibrinogen conformation in such a way as to render its crosslinking acceptor sites accessible to the action of F XIIIa,

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