scholarly journals Two functionally distinct pools of vitronectin (Vn) in the blood circulation: identification of a heparin-binding competent population of Vn within platelet alpha-granules

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 552-560 ◽  
Author(s):  
D Seiffert ◽  
RR Schleef
Keyword(s):  
Blood Circulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Heparin Binding ◽  
Immunoblotting Analysis ◽  
Sds Page ◽  
Altered Form ◽  
Activator Inhibitor

The biological functions of vitronectin (Vn) are dependent on its conformation. Whereas plasma Vn is present in a conformation that does not bind to heparin, platelet Vn has been recognized to be in a multimeric, conformationally altered form. To further understand the characteristics of platelet Vn, the molecules present in plasma and total and size-fractionated platelet releasates were compared (1) immunologically using three conformationally sensitive epitope-defined monoclonal antibodies, (2) functionally for their ability to interact with heparin, and (3) structurally using denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Our data indicate that Vn is present in platelet releasates in two molecular weight (M(r) forms. The high M(r) fractions contain conformationally and structurally altered Vn capable of interacting with heparin, and this form is distinct from plasma Vn and purified denatured Vn. In contrast, the lower M(r) forms of Vn are similar to plasma Vn. To determine if the presence of multimeric Vn requires platelet activation, platelets were disintegrated by sonication and fractionated by density gradients. Combined sodium dodecyl sulfate-PAGE (SDS-PAGE) and immunoblotting analysis showed a codistribution of multimeric Vn and type 1 plasminogen activator inhibitor in alpha-granule-rich fractions. Thus, platelet Vn is stored in a structurally and functionally distinct form from the molecule in plasma, raising the possibility that platelet- derived heparin-binding competent Vn will accumulate in areas of vascular injury.

Limited Proteolysis of Vitronectin by Plasmin Destroys Heparin Binding Activity

1991 ◽  
Vol 66 (03) ◽  
pp. 310-314 ◽  
Author(s):  
David C Sane ◽  
Tammy L Moser ◽  
Charles S Greenberg
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activator Inhibitor ◽  
Binding Activity ◽  
Binding Domain ◽  
Heparin Binding ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor ◽  
Pai 1

SummaryVitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN.Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.

scholarly journals Mechanism of protein C-dependent clot lysis: role of plasminogen activator inhibitor

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1218-1223 ◽  
Author(s):  
Y Sakata ◽  
DJ Loskutoff ◽  
CL Gladson ◽  
CM Hekman ◽  
JH Griffin
Keyword(s):  
Plasminogen Activator ◽  
Protein C ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Activated Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Species Specificity ◽  
Tissue Type ◽  
Clot Lysis ◽  
Activator Inhibitor

Abstract The mechanism by which activated protein C stimulates fibrinolysis was studied in a simple radiolabeled clot lysis assay system containing purified tissue-type plasminogen activator, bovine endothelial plasminogen activator inhibitor (PAI), plasminogen, 125I-fibrinogen and thrombin. Fibrinolysis was greatly enhanced by the addition of purified bovine activated protein C; however, in the absence of PAI, activated protein C did not stimulate clot lysis, thus implicating this inhibitor in the mechanism. In clot lysis assay systems containing washed human platelets as a source of PAI, bovine-activated protein C-dependent fibrinolysis was associated with a marked decrease in PAI activity as detected using reverse fibrin autography. Bovine-activated protein C also decreased PAI activity of whole blood and of serum. In contrast to the bovine molecule, human-activated protein C was much less profibrinolytic in these clot lysis assay systems and much less potent in causing the neutralization of PAI. This species specificity of activated protein C in clot lysis assays reflect the known in vivo profibrinolytic species specificity. When purified bovine-activated protein C was mixed with purified PAI, complex formation was demonstrated using immunoblotting techniques after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These observations suggest that a major mechanism for bovine protein C- dependent fibrinolysis in in vitro clot lysis assays involves a direct neutralization of PAI by activated protein C.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

2019 ◽  
Author(s):  
M Karunakaran ◽  
Vivek C Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S K Das ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heparin Binding ◽  
Sds Page ◽  
Significant Difference ◽  
Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312 ◽  
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

Abstract The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

scholarly journals Low-affinity heparin stimulates the inactivation of plasminogen activator inhibitor-1 by thrombin

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1164-1172 ◽  
Author(s):  
PA Patston ◽  
M Schapira
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Initial Increase ◽  
Plasminogen Activator Inhibitor 1 ◽  
Thrombin Inhibition ◽  
Zymographic Analysis ◽  
Activator Inhibitor ◽  
Pai 1

Abstract The influence of heparin on the reaction between thrombin and plasminogen activator inhibitor-1 (PAI-1) has been examined. With a 50- fold excess of PAI-1, the rate constant for the inhibition of thrombin was 458 mol/L-1s-1, which increased to 5,000 mol/L-1s-1 in the presence of 25 micrograms/mL unfractionated heparin or heparin with low affinity for antithrombin. The effect of low affinity heparin was then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using close to equimolar concentrations of reactants. Thrombin and PAI-1 formed a stable stoichiometric complex in the absence of heparin, which did not dissociate after the addition of 25 micrograms/mL low-affinity heparin. In contrast, when low-affinity heparin was added at the beginning of the reaction, there was an initial increase in PAI-1- thrombin complex formation, but this was rapidly followed by substantial proteolytic cleavage of unreacted PAI-1 and of the thrombin- PAI-1 complex. The idea that the relative concentrations of thrombin and PAI-1, and the presence of low affinity heparin, could influence the products of the reaction was examined in detail. Quantitative zymographic analysis of tissue plasminogen activator and PAI-1 activities and chromogenic substrate assay of thrombin activity showed that low-affinity heparin stimulated the inactivation of PAI-1 by an equimolar amount of thrombin, but caused only a minimal stimulation of thrombin inhibition. It is concluded that low-affinity heparin stimulates thrombin inhibition when PAI-1 is in excess, but, unexpectedly, that low-affinity heparin enhances PAI-1 inactivation when thrombin is equimolar to PAI-1.

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

2002 ◽  
Vol 76 (3) ◽  
pp. 217-223 ◽  
Author(s):  
J. Martinez ◽  
J. Perez-Serrano ◽  
W.E. Bernadina ◽  
F. Rodriguez-Caabeiro
Keyword(s):  
Alkaline Phosphatase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Differential Survival ◽  
Immunoblotting Analysis ◽  
Sds Page ◽  
Shock Proteins ◽  
Dose Dependent ◽  
Trichinella Nativa

AbstractStress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS–PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS–PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS–PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region

2020 ◽  
Vol 94 ◽  
Author(s):  
J.S. Dar ◽  
U. Shabir ◽  
S.A. Dar ◽  
B.A. Ganai
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ovis Aries ◽  
Diagnostic Specificity ◽  
Immunoblotting Analysis ◽  
Accurate Identification ◽  
North West ◽  
Dicrocoelium Dendriticum ◽  
Specificity And Sensitivity ◽  
Sds Page

Abstract Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as ‘small liver fluke’ disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS–PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS–PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

scholarly journals Mechanism of protein C-dependent clot lysis: role of plasminogen activator inhibitor

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1218-1223
Author(s):  
Y Sakata ◽  
DJ Loskutoff ◽  
CL Gladson ◽  
CM Hekman ◽  
JH Griffin
Keyword(s):  
Plasminogen Activator ◽  
Protein C ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Activated Protein C ◽  
Plasminogen Activator Inhibitor ◽  
Species Specificity ◽  
Tissue Type ◽  
Clot Lysis ◽  
Activator Inhibitor

The mechanism by which activated protein C stimulates fibrinolysis was studied in a simple radiolabeled clot lysis assay system containing purified tissue-type plasminogen activator, bovine endothelial plasminogen activator inhibitor (PAI), plasminogen, 125I-fibrinogen and thrombin. Fibrinolysis was greatly enhanced by the addition of purified bovine activated protein C; however, in the absence of PAI, activated protein C did not stimulate clot lysis, thus implicating this inhibitor in the mechanism. In clot lysis assay systems containing washed human platelets as a source of PAI, bovine-activated protein C-dependent fibrinolysis was associated with a marked decrease in PAI activity as detected using reverse fibrin autography. Bovine-activated protein C also decreased PAI activity of whole blood and of serum. In contrast to the bovine molecule, human-activated protein C was much less profibrinolytic in these clot lysis assay systems and much less potent in causing the neutralization of PAI. This species specificity of activated protein C in clot lysis assays reflect the known in vivo profibrinolytic species specificity. When purified bovine-activated protein C was mixed with purified PAI, complex formation was demonstrated using immunoblotting techniques after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These observations suggest that a major mechanism for bovine protein C- dependent fibrinolysis in in vitro clot lysis assays involves a direct neutralization of PAI by activated protein C.

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