2-Methylthio-Adenosine 5'-Diphospllate (2MeSADP), A High Affinity Probe for ADF Receptors on the Human Platelet

1979 ◽  
Author(s):  
D.E. Macfarlane ◽  
P.C. Srivastava ◽  
D.C.B. Mills
Keyword(s):  
Adenylate Cyclase ◽  
Binding Sites ◽  
Human Platelet ◽  
Shape Change ◽  
Order Rate Constant ◽  
Hill Coefficient ◽  
First Order ◽  
Affinity Probe ◽  
Binding Data ◽  
Specific Receptors

2MeSADP [β-32P] (6 ci/m mole) was synthesized and purified by HPLC. 2MeSADP aggregated platelets and inhibited the stimulation of adenylate cyclase by PGE1 with activities relative to ADP of 4 and 300 respectively. This selectivity for the cyclase site confirms the conclusion from other evidence that this site differs from the site that mediates shape change and aggregation. [B-32P] 2MeSADP bound rapidly (<20 sec) to platelets in PRP with half saturation at 18 nM and 450-500 binding sites per Platelet Under the same conditions PCE1-stimulated adenylate cyclase activity was 50% inhibited at 7 nM with a Hill coefficient of unity. With platelets in Tyrodes solution the apparent affinity was 2-4 nM. Other nucleotides competed for binding to washed platelets with the following Ki (μM):ADP, 3; ATP, 7;IDP, 150; GDP, 350 and AMP, 790. Binding was abolished by 100 μM p-chloromercuribenzene sulfonate, which blocks the inhibition of adenylate cyclase without affecting shape change. Bound 2MeSADP was completely displaced by excess ADP, with first order rate constant 1.54 mIn. These results, taken together with binding data for dlhydroergocryptine (Newman, et al JC1, 61: 395, 1978) and for prostacyclin (Siegl, et al, JCI, in press), suggest that the human platelet hae ca. 200-500 adenylate cyclase units per cell, each coupled to several specific receptors.

scholarly journals Kinetics of ADP-induced human platelet shape change: apparent positive cooperativity

1980 ◽  
Vol 58 (1) ◽  
pp. 45-52 ◽  
Author(s):  
John G. Milton ◽  
W. Yung ◽  
C. Glushak ◽  
M. M. Frojmovic
Keyword(s):  
Light Transmission ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Hill Coefficient ◽  
A Value ◽  
The Hill ◽  
Kinetics Of ◽  
Type Response ◽  
Platelet Shape

The kinetics of ADP-induced human platelet shape change have been examined. Initial velocities of platelet shape change were estimated by two methods: (1) the slope of the initial decrease in light transmission through stirred, citrated platelet-rich plasma, and (2) direct examination of platelet morphologies by phase-contrast microscopy. In both cases, a value of the Hill coefficient, NH, significantly greater than 1 is obtained (2.0 ± 0.2 and 1.8 ± 0.2, respectively). The observed elevated value of NH is not due to a substantial fraction of the ADP being platelet bound, the presence of factors in the plasma, platelet heterogeneity, or the influence of the rate of platelet shape change reversion. Our observations suggest that ADP-induced platelet shape change may be a positively cooperative or "threshold" type response.

scholarly journals ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'- p-fluorosulfonylbenzoyladenosine is a weak platelet agonist

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 751-756 ◽  
Author(s):  
AK Rao ◽  
MA Kowalska
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Binding Sites ◽  
Calcium Concentration ◽  
Shape Change ◽  
Ionized Calcium ◽  
Platelet Binding ◽  
Thiol Reagent ◽  
Dose Dependent ◽  
Platelet Shape

Abstract Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5′-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.

Proteolysis of the platelet surface: dissociation of shape change from aggregation

1986 ◽  
Vol 250 (4) ◽  
pp. H550-H557
Author(s):  
E. Kornecki ◽  
Y. H. Ehrlich ◽  
D. H. Hardwick ◽  
R. H. Lenox
Keyword(s):  
Adenylate Cyclase ◽  
Binding Sites ◽  
Prostaglandin E1 ◽  
Direct Evidence ◽  
Shape Change ◽  
Adenylate Cyclase System ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Induced Aggregation ◽  
Platelet Shape

Stimulation of intact platelets by ADP results in a shape change followed by aggregation in the presence of fibrinogen. ADP was found to induce a shape change in chymotrypsin-treated platelets that was similar in extent and initial velocity to that of intact (untreated) platelets. Scanning-electron microscopy verified an ADP-induced shape change in chymotrypsin-treated platelets. This shape change could be completely blocked by stimulators of platelet adenylate cyclase (forskolin, prostaglandin E1, and prostacyclin). On the other hand, the aggregation of chymotrypsin-treated platelets by fibrinogen was not dependent on the presence of ADP and could not be blocked by forskolin, prostaglandin E1, or prostacyclin, even though the levels of cyclic AMP (cAMP) formed in chymotrypsin-treated platelets were comparable to levels that completely inhibited the ADP-induced aggregation of intact platelets. This lack of inhibition of platelet aggregation was not due to degradation of the adenylate cyclase or prostaglandin receptors, since chymotrypsin-treated platelets were found to have a functional adenylate cyclase system that could be stimulated by forskolin, prostaglandin E1, and prostacyclin and inhibited by ADP and epinephrine, similar to that of intact platelets. These results provide direct evidence that cAMP does not interact with fibrinogen binding sites once they have become permanently exposed on the surface of platelets. Pretreatment of platelets with chymotrypsin therefore appears to be a useful tool that allows for the dissociation of platelet shape change from aggregation, without inhibiting either response.

The use of stable prostaglandins to investigate prostacyclin (PGI2)-binding sites and PGI2-sensitive adenylate cyclase in human platelet membranes

1984 ◽  
Vol 27 (2) ◽  
pp. 321-333 ◽  
Author(s):  
M. Lombroso ◽  
S. Nicosia ◽  
R. Paoletti ◽  
B.J.R. Whittle ◽  
S. Moncada ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Binding Sites ◽  
Human Platelet ◽  
Platelet Membranes

scholarly journals Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 140-148
Author(s):  
G Di Minno ◽  
P Thiagarajan ◽  
B Perussia ◽  
J Martinez ◽  
S Shapiro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Arachidonic Acid ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
High Affinity Site

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

Inactivation Of Purified Human Platelet Cyclic Amp Phosphodiesterase By The Affinity Label 2’-O-Iodohydrin-P-Cyclic Amp

1981 ◽  
Author(s):  
P G Grant ◽  
R F Colman ◽  
A K Sinha ◽  
R W Colman
Keyword(s):  
Cyclic Amp ◽  
Rate Constant ◽  
Active Site ◽  
Human Platelet ◽  
Order Rate Constant ◽  
Supernatant Fraction ◽  
First Order ◽  
Cyclic Amp Phosphodiesterase ◽  
Order Rate ◽  
Pseudo First Order

Cyclic AMP phosphodiesterase (PDE) is a regulatory enzyme in human platelets. Inhibitors of this enzyme raise intracellular cAMP which prevents platelet activation. Little is known about the biochemistry of this enzyme. PDE was isolated from human platelet concentrates by nitrogen bomb cavitation. The specific activity of PDE in cell lysate was 0.064 nmoles cAMP hydrolysed/min/mg protein at 22° , 1 µM cAMP. Eighty percent of the activity appeared in the 100,000 × g supernatant fraction. Chromatography was performed on DEAE cellulose equibrated with 50 mM Tris- acetate pH 6.0, 3.75 mM 2-mercaptoethanol. A linear gradient with a limiting salt concentration of 1.0 M Na acetate separated two peaks of PDE activity. The first had a for cAMP of >100 µM; the second had a Km for cAMP of 5 µM. The lower enzyme was further purified by adsorption on blue dextran Sepharose in 50 mM Tris pH 7.5, 2 mM MgCl2 followed by affinity elution with 1 mM cAMP in the same buffer. These steps resulted in a 1700 purification of the enzyme (113 nmoles/min/mg). The compound 2’-0-iodohydrin- p-cAMP (IH-cAMP) is a cAMP derivative with an alkylating side chain. Incubation of PDE with 5 mM IH-cAMP at 37° resulted in 88% inactivation of the enzyme at 15 hours, compared to a control, with a corrected pseudo-first-order rate constant of 0.144 h-1. When cAMP (100 µM) was included in the inactivation mixture the corrected pseudo-first-order rate constant decreased to 0.064 h-1l. Thus, a 20-fold excess of cAMP protected 56% against inactivation by IH- cAMP. The inhibition was not reversed by gel filtration of the inactivated enzyme which removed IH-cAMP. These results suggest that IH-cAMP reacts with the active site of PDE to irreversibly inactivate the enzyme. IH-cAMP should prove to be a useful tool in understanding the chemistry of the active site of this important enzyme.

scholarly journals The Number and Nature of ADP Receptors on Human Blood Platelets Determined with a Photoaffinity Label

1977 ◽  
Author(s):  
D.E. Macfarlane ◽  
D.C.B. Mills
Keyword(s):  
Binding Sites ◽  
Silicone Oil ◽  
Shape Change ◽  
Blood Platelets ◽  
Binding Data ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Derivatives Of ◽  
Scatchard Plots ◽  
Human Blood Platelets

ADP interacts with platelets to cause shape change, aggregation and inhibition of adenylate cyclase. Previous attempts to measure the binding of ADP to its receptor on intact platelets have been frustrated by the low ratio of specifically bound ADP to the ADP trapped in centrifuged pellets. Several 2-substituted derivatives of ADP have higher apparent affinities for the ADP receptor, improving this ratio. We have prepared 2-azido 5'-ADP from 2-chloroadenosine in 20% yield. It was more active than ADP as an antagonist of PGE1-induced elevation of cyclic AMP (Ki = 96 nM, cf ADP Ki = 760 nM) and also as an inducer of shape change and aggregation. 2-Azido [β32P]-5'-ADP (2.5 Ci/mmole) was prepared and used to study binding. Platelets were separated from plasma by centrifugation through silicone oil (1.023 gm/ml) in a modified Eppendorf centrifuge. Scatchard plots of the binding data were resolved into two linear components, one of zero affinity, corresponding to trapped plasma, measured independently with [14C] sucrose, and a high affinity component with apparent KD = 110-160 nM. There were 400-700 of these binding sites per platelet. 2-Azido 5'-ADP is photolysable with light at <310 nm, forming a reactive nitrene potentially suitable for photoaffinity labelling. 2-Azido [β32P]-5'-ADP was photolysed in the presence of washed platelets which were subsequently rewashed, solubilized and electrophoresed on SDS-polyacrylamide gradient slabs. Several peaks of radioactivity were observed in the high molecular weight region of the gel (ca 100-250 kD) one of which was less labelled in the presence of excess ADP or ATP to block access of the label to the receptor.

Detection Of High Affinity Fibrinogen Binding Sites On Human Platelets Stimulated By ADP: Comparison Of Two Methods Of Platelet Separation

1981 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Thomas A Morinelli ◽  
Elizabeth Kornecki
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Gel Filtration ◽  
Human Platelets ◽  
Binding Studies ◽  
Test Analysis ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
Specific Receptors

Binding of fibrinogen to specific receptors on human platelets exposed by ADP results in platelet aggregation. There are controversial data regarding classes and number of fibrinogen receptors, the values range from one to two classes and 1,000-80,000 receptors per platelet as reported in the literature. We have studied the interaction of fibrinogen with a) platelets washed by differential centrifugation according to Mustard and colleagues (washed platelets - WP) and with b) gel-filtered platelets (GFP). Platelet aggregation was studied with 100 μM ADP and with various concentration of fibrinogen. Maximal velocities of aggregation for WP and GFP were 81 and 47 units per min, respectively, and the Km values for fibrinogen calculated from the rate of aggregation were 0.9 × 10-7M for WP and 5.8 × 10-7M for GFP. The level of platelet fibrinogen released into the suspension from WP and GFP amounted to 2.4 μg and 15.0 μg per 10 9 platelets/ml, respectively, as measured by the staphylococcal clumping test. Analysis of 125I-fibrinogen binding data by the method of Scatchard and Feldman revealed 1,300 high affinity receptors (KD 3.2 × 10-8M) and 80,000 low affinity receptors (KD 5.6 × 10-5M) for WP. The binding of 125I-fibrinogen to GFP was greatly diminished. The number of fibrinogen receptors exposed by ADP on GFP and their binding affinity are under investigation in our laboratory. In conclusion, GFP were less sensitive to fibrinogen than were WP as shown in the aggregation and 125I-fibrinogen binding studies. It appears that the method of platelet separation is critical for the assessment of fibrinogen binding. Platelet activation and release of intact platelet fibrinogen during gel-filtration may interfere with the detection of high affinity fibrinogen binding sites.

scholarly journals ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'- p-fluorosulfonylbenzoyladenosine is a weak platelet agonist

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 751-756
Author(s):  
AK Rao ◽  
MA Kowalska
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Binding Sites ◽  
Calcium Concentration ◽  
Shape Change ◽  
Ionized Calcium ◽  
Platelet Binding ◽  
Thiol Reagent ◽  
Dose Dependent ◽  
Platelet Shape

Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5′-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.

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