Colchicine Uptake and Binding by Human Platelets

1975 ◽  
Vol 34 (03) ◽  
pp. 780-794 ◽  
Author(s):  
Dianne M Kenney ◽  
Francis C Chao ◽  
James L Tullis ◽  
Gail S Conneely
Keyword(s):  
Time Dependence ◽  
Incubation Period ◽  
Binding Sites ◽  
Silicone Oil ◽  
Cold Treatment ◽  
Human Platelets ◽  
Temperature Dependent

SummaryThe uptake and binding of antimitotic alkaloid colchicine has been demonstrated in washed preparations of human platelets. A silicone oil technique was adapted so that both uptake and binding of 14C-colchicine were examined in the same platelet preparations. The time dependence and amount of colchicine taken up and bound by different platelet preparations during a 90 to 120 min incubation period were highly reproducible. Both colchicine uptake and binding by intact platelets, and colchicine binding by preparations of lysed platelets were specific and temperature dependent. Colchicine uptake was slowly reversible. Magnesium and GTP enhanced colchicine binding by lysed platelet preparations but calcium decreased binding.Exposure of platelets to either cold (4° C) or to thrombin, which disrupt platelet microtubules, produced significant increases in colchicine uptake and binding. The thrombin effect was maximal at 37° C and resulted in a greater increase in uptake and binding than that produced by either cold treatment alone or, by cold treatment followed by incubation with thrombin at 37° C. The amount of increase in uptake and binding produced by thrombin was independent of both thrombin (1–5 Units/109 platelets) and colchicine concentrations (1–50 × 10−6M).It is postulated that thrombin may initiate the formation, or make available, colchicine binding sites (microtubule subunits) within platelets.

Relationship Between Aggregation and Binding of 125I-Fibrinogen and 45Calcium to Human Platelets

1979 ◽  
Author(s):  
E.I. Peerschke ◽  
R.A. Grant ◽  
M.B. Zucker
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Silicone Oil ◽  
Human Platelets ◽  
Mechanisms Of Action ◽  
Ionized Calcium ◽  
Clot Retraction ◽  
Fibrinogen Binding ◽  
Induced Aggregation ◽  
Stimulation Of

Since calcium and fibrinogen are essential cofactors for ADP-induced aggregation, their mechanisms of action were investigated. Aspirin-treated platelets were filtered through Sepharose 2B equilibrated with cation-poor Tyrode’s solution. After adding the radioactive compounds at 22, platelets were centrifuged through silicone oil. Trapping was assessed in separate samples with 14C-sorbitoi. Calcium binding was maximal at 1 hr and with 200 uH CaCl2. Two binding sites could be demonstrated on normal and thrombasthenic platelets and on platelets which had lost their ability to aggregate (but not to change shape or promote clot retraction) after treatment with EDTA (8 min, 37°, pH 7.8). ADP did not alter calcium binding in the presence or absence of fibrinogen. Fibrinogen, however, bound to normal gel filtered platelets in the presence of ADP and ionized calcium “ but not to thrombasthenic or EDTA-treated platelets or to normal platelets in the presence of EDTA or at pH 6.5. Binding of fibrinogen increased with concentration but saturation was not observed even at physiologic levels. Fibrinogen binding was similar in gel filtered platelets and citrated piatelet-rich plasma. These studies indicate that stimulation of platelets with ADP under conditions favorable to aggregation is associated with binding of fibrinogen but not of additional calcium.

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets

1994 ◽  
Vol 267 (6) ◽  
pp. C1543-C1552 ◽  
Author(s):  
M. Kimura ◽  
K. Nakamura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
J. P. Reeves ◽  
...  
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Free Medium ◽  
Amidolytic Activity ◽  
Cytosolic Ph ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

ACTIVATION DEPENDENT ALTERATIONS IN THE CHEMICAL CROSSLINKING OF ARGINYL-GLYCYL-ASPARTIC ACID (RGD) PEPTIDES WITH PLATELET GLYCOPROTEIN (GP) GPIIb-IIIa

1987 ◽  
Author(s):  
S E D’Souza ◽  
M H Ginaberg ◽  
S Lam ◽  
E A Plow
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Amino Acid Sequences ◽  
Platelet Glycoprotein ◽  
Chemical Crosslinking ◽  
Dependent Manner ◽  
Rgd Peptides ◽  
Dependent Inhibition ◽  
Adhesive Proteins ◽  
Dose Dependent

The platelet adhesive proteins, fibrinogen, fibronectin and von WillebrandFactor, contain RGD amino acid sequences; RGD-containing peptides inhibit the binding of these adhesive proteins to platelets; and a membrane receptor for these adhesive proteins binds to Arg-Gly-Asp and contains GPIIb-IIIa. The present study was undertaken to characterize the interaction of RGDpeptides with GPIIb-IIIa using a chemical crosslinking approach. A radioiodinated RGD-containing heptapeptide was bound to washed human platelets under conditions at which ≥ 85% of theinteraction was inhibited by excess nonlabeled peptide. After binding of the peptide to platelets for 45 min at22°, a homobifunctional crosslinking reagent was added, and the platelets were extracted and analyzed on polyacrylamide gels. With resting platelets,autoradiography of the gels revealedthat the peptide crosslinked tobothGPIIb and GPIIIa. This interaction wasinhibited by excess nonlabeled peptide but not by certain conservatively substituted RGD peptides. Stimulation of the platelets caused a dramatic increase in crosslinking of the peptide to only one of the two subunitsof GPIIb-IIIa. The stimulus dependentincrease in the crosslinking reactionwas specific and saturable as it was inhibited by RGD peptides in a dose dependent manner. In addition, peptides corresponding in structure to the carboxy terminus of the γ chain of fibrinogen also produced concentration dependent inhibition of the interaction. The increase in crosslinking induced by platelet stimulation was divalent ion dependent. Similar results werealso obtained with a second, larger RGD-containing peptide and with asecond chemical crosslinking reagent.Theseresults indicate that platelet stimulation in the presence of divalent ions causes a change which permitsmoreefficient crosslinking of RGD-containing peptides to only one of the two subunits of GPIIb-IIIa. The results are also compatible with a proximalrelationship of both subunits tothe RGD binding sites on the plateletmembrane.

The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets

1979 ◽  
Author(s):  
H.R. Gralnick ◽  
D.K. Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Scatchard Analysis ◽  
Human Fibrinogen ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

scholarly journals Antagonism by dibutyryl adenosine cyclic 3',5'-monophosphate and testololactone of concanavalin A capping.

1975 ◽  
Vol 66 (2) ◽  
pp. 392-403 ◽  
Author(s):  
B Storrie
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Cold Treatment ◽  
Intracellular Camp ◽  
Camp Concentration ◽  
Con A ◽  
Cell Rounding ◽  
Intracellular Camp Concentration

Exposure of CHO-K1 cells in vitro to dibutyryl adenosine cyclic 3',5'-monophosphate (DBcAMP) plus testololactone produces a rapid, reversible antagonism of ligand-induced collection of initially dispersed concanavalin A (Con A) binding sites into a caplike mass. Morphologically, as Con A capping occurs, the cells become less spread and then round completely. With prolonged Con A exposure, cells cultured in either the absence or the presence of DBcAMP plus testololactone cap and round. Capping is blocked by cold treatment and respiratory inhibitors. Colcemid at concentrations greater than 1 muM promotes both Con A capping and cell rounding. Cytochalasin B at similar concentrations inhibits both capping and cell rounding. Treatment of cells with Con A has little effect on intracellular cAMP concentration. Possible mechanisms by which cAMP may modulate the movement of Con A binding sites are discussed.

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

scholarly journals Affinity for (3H)Iloprost Binding Sites and cAMP Synthesis Activity of a 3-Oxa-methano Prostaglandin I1 Analog, SM-10906, in Human Platelets and Endothelial Cells.

1997 ◽  
Vol 74 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Takashi Hiroi ◽  
Keiko Maruyama ◽  
Kaoru Hattori ◽  
Toshio Ohnuki ◽  
Takafumi Nagatomo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Human Platelets ◽  
Camp Synthesis ◽  
Synthesis Activity
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