Flow Cytometry of Intracellular Calcium in Platelets

Textbook, 2012

30 Pages



The Role of Calcium in Intracellular Signal Transduction
Sources of Calcium
Signal Transduction
Activation of Platelets
Shape Change
Procoagulant Activity
Soluble Platelet Agonists
Inhibition of Platelets by cAMP

Flow Cytometry
Monoclonal Antibodies
Isotype Control
Monoclonal Antibodies Used for the Flow Cytometric Characterization of Platelets
Intracellular Probes
Presentation and Analysis of Data
Dotplots and Identification of Cells
Further Analysis
Regions and Gates

The Use of Fluo-3 as Intracellular Calcium Indicator
Brief Introduction

Flow Cytometric Analysis






During the last years flow cytometry has been developed into an important diagnostic tool with a wide range of applications. Whereas the differentiation of leukocytes is most often encountered, flow cytometry also renders itself especially useful in the study of platelets, . Commonly, surface markers are of key interest, but the fact that platelets can be conveniently activated in vitro offers the opportunity to investigate intracellular signal transduction processes, which lead to the release of calcium into the cytosol. And it is this rise of intracellular calcium that flow cytometry is able to detect in real time through the use of suitable calcium-sensitive markers.

It is the aim of the following chapters to put together an overview of this technique and therefore to provide a sound starting ground for its application to clinical and experimental investigations and also for its further development.

The Role of Calcium in Intracellular Signal Transduction

Much evidence has been brought together supporting the concept that calcium is the key second messenger in intracellular signal transduction of platelets. The increase of cytosolic free calcium represents a pivotal step during activation . It is accepted that the graded response of platelets to stimuli of varying intensity reflects a progressive increase in free calcium levels with a clear relationship between the intensity of the stimulus, the intracellular calcium concentration, and the intensity of the effects induced by the stimulus . These effects, triggered at distinct calcium levels, include processes like shape change, adhesion, secretion and aggregation of the platelets.

Sources of Calcium

The intracellular calcium concentration of resting platelets lies in the order of 40 to 100 nmol/l . Compared to the calcium concentration in the extracellular environment (i.e. in blood plasma) of roughly 2.5 mmol/l this represents a very steep concentration gradient, which is maintained predominantly by the action of a Ca2 +/Mg2 + ATPase pumping the calcium mainly into the dense tubular system and perhaps outside the cell .

Upon stimulation, the concentration increases to 2 to 10 µmol/l. Calcium is released from the main storage sites, namely the dense tubular system and the plasma membrane. The dense tubular system consists of membrane-limited tubules derived from the endoplasmatic reticulum of the megakaryocytes .

Depending on the agonist, considerable amounts of calcium can be brought to the cytosol from the extracellular fluid across the plasma membrane through specific calcium channels. Additionally, calcium is located in mitochondria and dense bodies (storage granules containing serotonin among other substances), but these sites are of little importance in the activation of the platelets .

Immediately after stimulation, the calcium concentration begins to revert to basal levels indicating that the calcium is redistributed to the storage sites, mainly the dense tubular system . This is achieved by the aforementioned calcium pump, which is activated through phosphorylation by a cAMP-dependent protein kinase .

Signal Transduction

Signal transduction pathways are shown in Figure 1. Platelet agonists like thrombin, ADP, collagen, and epinephrine, interact with their specific receptors on the cell surface. Agonist receptor interaction stimulates the enzyme phospholipase C via G-proteins. The activated phospholipase C cleaves PIP2 (phosphatidylinositol 4,5-bisphosphate) to IP3 (inositol 1,4,5- trisphosphate) and DAG (diacylglycerol). While the water-soluble IP3 enters the cytosol, the lipid-soluble DAG remains bound to the plasma membrane. Within milliseconds after receptor occupancy, IP3 induces the release of calcium from the storage sites . The mechanism of this release is not completely known, although some evidence exists that it is inhibited by calcium channel blockers .

In addition, the agonist-receptor interaction can lead to a rapid influx of calcium from the extracellular medium. It has been suggested that this process is governed by receptorcontrolled calcium channels or alkalinization of the cytosol .

DAG, the second product of the cleavage of PIP2 activates protein kinase C (PKC), which phosphorylates various proteins finally leading to the secretion of the contents of the storage granules. This DAG-PKC system can work independently from other signal transduction pathways, however, the efficiency is markedly enhanced by the presence of increased levels of free calcium .

Figure 1. Principal pathways of signal transduction upon stimulation leading to the activation of platelets. PIP2 - phosphatidylinositol 4,5-bisphosphate); IP3 - inositol 1,4,5-trisphosphate; DAG - diacylglycerol; TXA2 - thromboxane A2.

High concentrations of cytosolic free calcium are not only able to prepare the platelets directly for the formation of a haemostatic plug but can also induce a further signal transduction pathway by activating phospholipase A2. This enzymes uses membrane phospholipids like phosphatidylcholine and phosphatidylethanolamine as substrates for the formation of arachidonic acid, which is subsequently converted by cyclooxygenase to cyclic endoperoxides finally resulting in the generation of thromboxane A2 (TXA2). This substance is by itself a potent platelet agonist and induces vasoconstriction . Inhibitors of the cyclooxygenase, like acetylsalicylic acid and other non-steroidal anti-inflammatory drugs (NSAID), reduce the formation of TXA2. In the case of acetylsalicylic acid, the resulting decrease of platelet reactivity is used especially in the therapy of atherosclerotic diseases to reduce the risk of thrombus formation . With regard to other NSAIDs this mechanism represents a potential side effect .

Activation of Platelets


Endothelial cells maintain a non-thrombogenic surface of the vessel walls to prevent the initiation of haemostasis. Damage to the endothelial cell layer due to mechanical injury or degenerative processes results in the exposure of the normally covered subendothelial matrix. The various constituents of the subendothelium (e.g. collagen) are highly thrombogenic in terms of both platelet and coagulation cascade activation . Under conditions of high shear stress, which are present especially in arterioles (10 to 50 µm in diameter) and at stenoses of vessels, soluble von Willebrand Factor (vWF) adheres particularly to collagen and heparin-like glycosaminoglycanes. Initially, platelets interact with this form of bound vWF via the Gp Ib-IX complex, but as the high dissociation rate of this interaction is not able to bind the platelets firmly to the site of vessel injury, the platelets keep moving on the surface of the subendothelium in a rolling fashion, always maintaining the interaction between vWF and Gp Ib-IX. This results in a reduction of the velocity of the flowing platelets. The rolling process induces the activation of the platelets leading to a conformational change in the extracellular portions of glycoprotein IIb-IIIa (Gp IIb-IIIa) via the signal transduction pathways mentioned above .

Gp IIb-IIIa is a heterodimeric transmembrane molecule composed of an a and a b subunit (aIIbb3) belonging to the integrin receptor family together with several other glycoproteins . The activated form of Gp IIb-IIIa is able to bind irreversibly to vWF completing the adhesion process of the platelet .

In parts of the circulation with low shear stress, the initiation of haemostasis relies at least on two different types of platelet-vessel wall interaction. Platelets bind to collagen mediated by the collagen receptor which is a heterodimer designated Gp Ia-IIa (a2b1). On the other hand, Gp IIb-IIIa receptors of resting platelets recognize and adhere to surface-bound fibrinogen which has undergone a conformational change, whereas soluble fibrinogen can only interact with Gp IIb-IIIa of activated platelets. Both mechanisms finally result in the activation of the platelets .

Shape Change

In the course of platelet activation, the cells lose their normal (i.e. resting) discoid shape to appear as spheres with extending pseudopodia due to the increased polymerization of actin filaments and their association with myosin resulting in an increase of the surface area .


Through mediation of soluble fibrinogen interacting with activated Gp IIb-IIIa receptors, platelets form larger aggregates, a process which is thought to occur mainly under conditions of turbulent flow providing intensive contact between the cells. Non-turbulent conditions are suggested to support the formation of a monolayer of cells .

Monoclonal antibodies like the antibody fragment c7E3 (abciximab) directed against Gp IIb- IIIa significantly reduce aggregation, which is now clinically used to prevent the formation of thrombi especially after coronary angioplasty procedures . Studies have shown, however, that these antibodies are also able to reduce an agonist-induced calcium influx, suggesting that this glycoprotein may be involved in the calcium transport across the plasma membrane .


Apart from adhesion, shape change and aggregation another process, namely the secretion of storage granule contents, plays an important role in the formation of a haemostatic plug. α-granules constitute one third to one half of the platelet granule population with a diameter of 300 to 500 nm. They contain numerous secretory proteins of which fibrinogen and vWF are the most important. Dense bodies, whose name is derived from their dense (i.e. dark) appearence on electron-microscopic imaging, provide especially ADP, calcium and serotonin .

Secretion occurs when the concentration of intracellular calcium reaches a certain level which is higher than the level necessary for the induction of shape change and the activation of Gp IIb-IIIa receptors28. The substances are released into the extracellular medium by fusion of the membrane-limited granules with the outer plasma membrane and partly with the open canalicular system, which represents invaginations of the plasma membrane. Once in the extracellular medium, the released substances contribute to the activation of further platelets and promote the overall process of haemostasis .

Procoagulant Activity

With regard to the coagulation system, platelets do not restrict themselves to the interaction with fibrinogen via their Gp IIb-IIIa receptors in order to form the haemostatic meshwork of cells and fibrin strands. Actually, platelets provide an ideal surface to enhance a number of steps in coagulation: By exposing negatively charged phospholipids during activation, platelets bind the constituents of the prothrombinase complex (factor Va, factor Xa and calcium) which catalyzes the conversion of prothrombin to thrombin; the tenase complex (factor VIIIa, factor IXa and calcium) promotes the activation of factor X; vWF, which interacts especially with Gp IIb-IIIa, is associated with factor VIII .

Soluble Platelet Agonists

In addition to the activation of platelets by interaction with the subendothelium, several soluble agents are able to stimulate platelets, most of them acting via G-protein-linked receptors.

ADP (adenosine diphosphate), which appears in plasma only through dense body secretion and lysis of blood cells, induces an increase in the cytosolic calcium concentration mainly via an influx of calcium across the plasma membrane and only to a lesser extent through the generation of IP3 and the subsequent release of calcium from the dense tubular system. ADP is able to act synergistically with thromboxane A2 (TXA2). As described above (see section Signal Transduction), TXA2 is produced intracellularly during platelet activation in the course of the arachidonic acid metabolism. To get into the extracellular fluid, it passes directly through the plasma membrane, where it supports the activation of further platelets by interacting with a specific TXA2 receptor .

Thrombin, the generation of which is greatly amplified by platelets themselfes (see above), is a main product of the plasmatic coagulation cascade and converts fibrinogen to fibrin . At the same time, thrombin is a potent platelet activator of central importance under physiological conditions. It induces the whole spectrum of activation effects and is able to act independently from the formation of TXA2. Thrombin mobilizes calcium from internal and external stores .

Even though epinephrine activates platelets only at unphysiologically high concentrations, it is an interesting feature that the interaction of epinephrine with a2 receptors of the platelet initiates activation only in the presence of extracellular calcium. The process seems to be completely dependent on an increased transmembrane permeability for calcium. Aggregation and secretion, but no shape change is induced,.

Inhibition of Platelets by cAMP

Whereas calcium is the pivotal intracellular messenger in terms of platelet activation, cAMP (cyclic adenosine monophosphate) plays a preeminent role in inhibiting platelet function . Certain substances, especially the prostaglandins PGI2 (also referred to as prostacyclin, mainly produced by endothelial cells) and PGE1, inhibit platelet reactivity by raising the intracellular cAMP level through the stimulation of adenyl cyclase. Phosphodiesterase inhibitors exert a similar effect by reducing the activity of phosphodiesterase, an enzyme responsible for the degradation of cAMP.

cAMP decreases agonist binding to receptors, inhibits the generation of activating signal molecules in the phosphoinositide pathway and reduces the intracellular calcium concentration by activating a pump which sequestrates calcium from the cytosol mainly into the dense tubular system. These mechanisms result in the reduction of the magnitude and duration of calcium transients leading to decreased platelet reactivity. It is noteworthy that all physiologic agonists inhibit adenyl cyclase but the inhibition of this enzyme alone is not sufficient to trigger platelet activation.


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Flow Cytometry of Intracellular Calcium in Platelets
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flow, cytometry, intracellular, calcium, platelets
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Thomas Gamsjäger (Author), 2012, Flow Cytometry of Intracellular Calcium in Platelets, Munich, GRIN Verlag, https://www.grin.com/document/190163


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