SUB-SECOND CALCIUM DYNAMICS IN ADP AND THROMBIN-STIMULATED PLATELETS; ASSESSED BY A C0NTINU0US-EL0W APPROACH

1987 ◽  
Author(s):  
A R L Gear ◽  
G D Jones
Keyword(s):  
Protein Phosphorylation ◽  
Shape Change ◽  
Reaction Times ◽  
Human Platelets ◽  
Free Calcium ◽  
Calcium Dynamics ◽  
On Line ◽  
Micro Flow ◽  
Flow Apparatus ◽  
Emission Changes

There is now evidence that many platelet reactions begin within 1 sec of platelet stimulation. These include "shape change," aggregation and biochemical events such as protein phosphorylation. Our laboratory has devised quenched-flow approaches for following such early events (J Lab Clin Med 100, 866, 1982) and we have extended these to fluorimetric analyses of rapid calcium changes. A micro, flow-through cell, with a sensing volume of 0.1 μ1, is placed on line from the quenched-flow apparatus. Indo-1 loaded, human platelets are pumped through the system and reaction times from 0.25 sec can be followed. Ratioing emission changes at 400 and 480 nm, after excitation at 355 nm, provides an index of free calcium. ADP (10 μM) induced a rapid increase in Ca++ to about 1 μM by 1.5 sec, beginning near 0.3 sec. This was faster and greater than the first increase caused by thrombin (10U/ml). However, thrombin induced a second (> 5s) and larger increase in free platelet calcium. Control experiments where the Indo-1 loaded platelets were simply pumped through the 0.3 mm ID reaction tubing, revealed a slight increase above resting calcium values, indicating some shear-induced activation. The use of the continuous-flow fluorescent cell coupled to the quenched-flow apparatus enables following calcium dynamics under Theological conditions very close to those iui vivo.Correlations with other early events, such as protein phosphorylation, become possible. Supported by NIH HL-27014.

ADRENALINE ACTIVATES HUMAN PLATELETS BUT IS NOT PER SE AN AGGREGATING AGENT. EFFECTS ON PLATELET MORPHOLOGY, MEMBRANEFLUIDITY, FIBRINOGEN BINDING, CYTOPLASMIC FREE CALCIUM AND PROTEIN PHOSPHORYLATION

1987 ◽  
Author(s):  
M Lanza ◽  
A Beretz ◽  
A Stierlé ◽  
D Hanau ◽  
M Kubina ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Membrane Fluidity ◽  
Direct Effect ◽  
Shape Change ◽  
Human Platelets ◽  
Free Calcium ◽  
Blood Collection ◽  
Fibrinogen Binding ◽  
Per Se

Adrenaline (Adr) is generally considered as a full agonist able to induce in vitro the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. Adr 2.5 M) induces the aggregation and secretion of 41 % of preloaded 3H-serotonin in human platelets in citrated plasma. This effect is not seen in plasma collected on 50 ATU/ml hirudin, and is due to the generation of traces of thrombin during blood collection and not to a direct effect of citrate itself, such asthe lowering of plasma free calcium. With washed human platelets suspended in Tyrode's buffer containing 2 mM Ca2+, 0.35 %albumin and apyrase, Adr (0.1 -100 M) doesnot cause shape change, aggregation or secretion of serotonin and does not modify platelet ultrastructure as judged by electron microscopy. Adr (1-100 M) does not change platelet membrane fluidity, as studied with the lipophilic fluorescent probe TMA-DPH. Adr has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+levels measured with quin2, or phosphorylation of 20 KDa or 47 KDapolypeptides, whereas all these parameters are modified after stimulation with ADP orthrombin. Adr potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels,membrane fluidity, fibrinogen binding or protein phosphorylation. This effect is also seen with alpha2-adrenergic agonists (noradrenaline, alpha-methyl noradrenaline, clonidine) and is inhibited by alpha2-adrenergicantagonists such as yohimbine. The potentiation of platelet aggregation by Adr is not modified by prior incubation of the platelets with1mM aspirin for 15 min. This study shows that Adr alone does not induce modifications ofmorphology, metabolism or function of intactand functional washed human plateletsand that Adr cannot be considered per se as an aggregating agent. However, Adr interactswith alpha2-adrenergic receptors on human plateletsand potentiates biochemical and aggregatory responses induced by other platelet agonists.

Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

1987 ◽  
Vol 58 (02) ◽  
pp. 737-743 ◽  
Author(s):  
Frarnçois Lanza ◽  
Alain Beretz ◽  
Martial Kubina ◽  
Jean-Pierre Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fluorescent Indicators ◽  
Fura 2 ◽  
Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

scholarly journals Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1539-1543
Author(s):  
GD Jones ◽  
AR Gear
Keyword(s):  
Blood Flow ◽  
Continuous Flow ◽  
Blood Platelets ◽  
Adenosine Diphosphate ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Dynamics ◽  
Flow Conditions ◽  
Shear Rates ◽  
Flow Apparatus

The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single- particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).

Epinephrine potentiates human platelet activation but is not an aggregating agent

1988 ◽  
Vol 255 (6) ◽  
pp. H1276-H1288 ◽  
Author(s):  
F. Lanza ◽  
A. Beretz ◽  
A. Stierle ◽  
D. Hanau ◽  
M. Kubina ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Membrane Fluidity ◽  
Direct Effect ◽  
Shape Change ◽  
Human Platelets ◽  
Blood Collection ◽  
Fibrinogen Binding ◽  
Serotonin Secretion

Epinephrine can in certain in vitro conditions induce the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. This study examines some functional and biochemical responses to epinephrine. Epinephrine induces the aggregation and serotonin secretion of human platelets in citrated plasma. This is not due to a direct effect of citrate itself, such as the lowering of plasma free Ca2+ but more likely to the generation of traces of thrombin during blood collection, as suggested by abrogation of these platelet responses when hirudin was added before citrate. When washed human platelets suspended in Tyrode buffer containing 2 mM Ca2+, 0.35% albumin and apyrase, and 0.1-100 microM epinephrine were used, no shape change, aggregation, or secretion of serotonin was observed, nor was the platelet ultrastructure modified. Epinephrine does not modify platelet membrane fluidity, as studied with the lipophilic fluorescent probe trimethylammonium-diphenylhexatriene. It has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+ levels measured by quin2, or protein phosphorylation. Epinephrine potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels, membrane fluidity, fibrinogen binding, or protein phosphorylation. These effects are mediated by alpha 2-adrenergic agonists and inhibited by alpha 2-adrenergic antagonists. This study shows that epinephrine alone does not induce modifications of morphology, metabolism, or function of intact and functional washed human platelets and that it cannot be considered per se as an aggregating agent. However, epinephrine interacts with alpha 2-adrenergic receptors on human platelets and potentiates biochemical and aggregatory responses induced by other platelet agonists.

scholarly journals Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1539-1543 ◽  
Author(s):  
GD Jones ◽  
AR Gear
Keyword(s):  
Blood Flow ◽  
Continuous Flow ◽  
Blood Platelets ◽  
Adenosine Diphosphate ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Dynamics ◽  
Flow Conditions ◽  
Shear Rates ◽  
Flow Apparatus

Abstract The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single- particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).

scholarly journals Activation of human platelets by the rabbit anticardiolipin antibodies

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3135-3143 ◽  
Author(s):  
YL Lin ◽  
CT Wang
Keyword(s):  
Solid Phase ◽  
Shape Change ◽  
Human Platelets ◽  
Anticardiolipin Antibodies ◽  
Ion Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Free Calcium ◽  
Dependent Manner ◽  
Platelet Response ◽  
Inhibition Studies

Abstract Affinity purified anticardiolipin antibodies (ACLA) raised in rabbits showed cross-reactivities with various negatively charged phospholipids as shown by both the solid phase enzyme-linked immunosorbent assay (ELISA) and inhibition studies. In ELISA, ACLA showed strong cross-reactivity to both sphingomyelin (SM) and phosphatidylethanolamine (PE), but the inhibition studies showed that ACLA failed to bind the aqueous suspensions of SM, PE, and PE/PC (1:1). ACLA bound to resting gel-filtered human platelets (GFP) as shown by both inhibition study and flow cytofluorometric analysis. Western blotting procedure showed that ACLA strongly cross-reacted to an 80-Kd plasma membrane protein. ACLA activated platelet response in a concentration-dependent manner. At less than 10 micrograms/mL, ACLA induced both platelet shape change to spiculate irregular forms as shown by scanning electron microscopy and the phosphorylation of 20-Kd protein. ACLA at more than 10 micrograms/mL caused platelet aggregation and secretion. The aggregation was inhibited by EDTA; aspirin; antimycin A plus 2-deoxyglucose; PGE1; and the F(ab')2 fragment of ACLA. It was not inhibited by monoclonal antibody to Fc receptor (MoAb FcR2). The biochemical events of ACLA-induced platelet response involved the elevation of (1) thromboxane A2 formation, (2) cytosolic free calcium ion concentration ([Ca2+]i), and (3) 47-Kd protein phosphorylation. In addition, the subaggregatory concentration of ACLA showed synergistic platelet activation with that concentration of thrombin, collagen, and epinephrine. The study showed the mechanism involved in ACLA-induced platelet responses.

INCREASED AGGREGATION AND SECRETION RESPONSES OF HUMAN PLATELETS WHEN LOADED WITH THE CALCIUM FLUORESCENT PROBES QUIN2 AND FURA-2

1987 ◽  
Author(s):  
F Lanza ◽  
A Beretz ◽  
M Kubina ◽  
J-P Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fura 2 ◽  
Low Concentrations ◽  
Platelet Shape

Incubation of human platelets with the fluorescent dye esters quin2-AM (10 μM) or fura-2-AM (1 μM) makes possible the direct measurement of intracellular free calcium ([Ca2+1).Underthese conditions, basal levels of [Ca2+]i of 120 ± 16 nM (n=23) using quin2 and 137 ± 15 nM (n=5) using fura-2 can be measured. Both probes record comparable increases of [Ca2 ]i after stimulation with ADP, thrombin, PAF, or U-46619. Incorporation into human platelets of quin2 or fura-2 at the concentrations used to monitor [Ca2+]i leads to the activation of platelets. This was shown by increased aggregation and secretion responses of quin2or fura-2 loaded platelets after stimulationwith ADP (5 μM), PAF (1 μM) and with low concentrations of thrombin (0.015U/ml), collagen (0.5 μg/ml), the endoperoxide analog U-46619 (0.5 μM) or the calcium ionophore A 23187 (1 μM). Quin2 and fura-2 mediated platelet activation could be due to altered arachidonic acid metabolism, since it was partly inhibited by prior treatment with the cyclooxygenase inhibitor acetylsalicylate (1 μM). In contrast, platelets loaded with higher concentrations of calcium chelators (20 to 100 μM quin2-AM)exhibited diminished aggregation responses to all aggregating agents. Thislatter effectwas accompanied by increased fluidity of theplatelet plasma membrane bilayer and by the exposure of a new pool of membranes at the outer surface of platelets, as monitored withtrimethylammonium-diphenylhexatriene (TMA-DPH) in platelets loaded with thenon-fluorescent calcium probe analog MAPT. Platelet shape change, as measured in the aggregometer, was dose-dependently inhibited after loading of quin2 (10-50 μM quin2-AM), even at concentrations which potentiated aggregation. We conclude that incorporation of intracellular calcium chelators alters platelet responses, including at concentrations used to monitor intracellular calcium changes.

Mechanism of Action of a Potent Antiplatelet Peptide, Triflavin from Trimeresurus flavoviridis Snake Venom

1991 ◽  
Vol 66 (04) ◽  
pp. 489-493 ◽  
Author(s):  
Tur-Fu Huang ◽  
Joen-Rong Sheu ◽  
Che-Ming Teng
Keyword(s):  
Snake Venom ◽  
Shape Change ◽  
Membrane Surface ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Free Calcium ◽  
Activation Process ◽  
Trimeresurus Flavoviridis ◽  
Fibrinogen Binding ◽  
Induced Aggregation

SummaryTriflavin, an antiplatelet peptide from Trimeresurus flavoviridis snake venom, inhibits aggregation of human platelets stimulated by a variety of agonists. However, triflavin does not affect the shape change and release reaction of platelets stimulated by thrombin and collagen. In this paper, we further investigate its effect on the intracellular events occurring after the activation of platelets. Triflavin does not inhibit the intracellular free calcium rise of Quin 2-AM loaded platelets stimulated by thrombin and it also has no significant effect on thromboxane B2 formation of platelets stimulated by thrombin. Triflavin does not affect the 3(H)-inositol monophosphate formation of the 3(H)-myoinositol loaded platelets. However, triflavin dose-dependently inhibits fibrinogen-induced aggregation and 125I-fibrinogen binding of ADP-stimulated platelets. In addition, triflavin dose-dependently blocks fibrinogen-induced aggregation of elastase-treated platelets. It is concluded that triflavin specifically inhibits fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex on platelet membrane surface without any inhibitory effect on the platelet-activation process.

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