Laboratory Markers of Platelet Activation

Paul Harrison

Alan D. Michelson

Prevention of platelet activation in the intact circulation is normally controlled by the antithrombotic nature of the endothelial cell surface, which exhibits a number of effective mechanisms for inhibiting platelets. Under normal conditions, platelets are therefore unable to form stable contacts on the vessel wall. Platelet adhesion, however, can occur upon vessel wall damage, on diseased/activated endothelial cells, or at sites of disturbed flow (e.g., atherosclerotic plaques). Exposure of the subendothelial matrix (e.g., collagen) initiates rapid platelet activation, which is then further amplified by the release of platelet-derived adenosine diphosphate (ADP), thromboxane, and serotonin, resulting in further platelet activation and recruitment of other platelets in the vicinity to rapidly form a hemostatic plug. The high shear stresses associated with mature atherosclerotic lesions can also initiate platelet activation.

Platelet activation results in a complex series of changes including a physical redistribution of receptors, changes in the molecular conformation of receptors, secretion of granule contents, cleavage and release of various glycoproteins (the “sheddome”), generation of thromboxane, development of a procoagulant surface, generation of platelet-derived microparticles, and formation of both platelet-platelet and leukocyte-platelet aggregates. All of these changes can potentially be used as markers of platelet activation (Table 67.1). Although platelet activation is usually initiated locally at the site of formation of the hemostatic plug or thrombus, it is possible to measure the consequences of these local events in the systemic circulation—in part because thrombi are very dynamic with transient adhesion, “rolling,” and embolization of platelets often occurring. Also, in thrombotic disease, platelets may be “primed” or more reactive to agonists in the circulation or conversely “exhausted” (due to excessive activation) and less activatable ex vivo. This can be augmented by a genetic predisposition causing a preexisting reactive phenotype. However, individuals may also acquire a more reactive phenotype due to many environmental phenotypic factors including diet, life-style, ageing, various diseases, etc. Therefore, it is now possible to utilize a number of sensitive tools to measure inappropriate platelet activation and/or reactivity in health and disease and also to monitor various forms of antiplatelet therapy to potentially modify treatment and prevention strategies.

Whole blood flow cytometry¹ is now regarded as the research method of choice for the measurement of all these changes, except the secretion of soluble molecules and shed glycoproteins, which are now usually measured by ELISA. Whole blood flow cytometry has many advantages: (a) minuscule volumes (˜5 µL) of blood can be used; (b) platelets are directly analyzed in their physiologic milieu of whole blood; (c) the minimal manipulation of samples prevents artifactual in vitro activation and potential loss of platelet subpopulations; (d) both the activation state of circulating platelets and the reactivity of circulating platelets can be determined; and (e) a spectrum of different activation-dependent changes can be determined.

METHODS AVAILABLE TO MEASURE PLATELET ACTIVATION

Biochemical Markers

Thromboxane generation provides one of the most important amplification loops in platelet biochemistry. This is exemplified by the widespread use of aspirin to target cyclooxygenase (COX)-1, resulting in irreversible inhibition of thromboxane A₂ generation for the lifespan of the platelet and partially explains why aspirin is such an effective antithrombotic therapy. Serum, plasma, and urinary assays for thromboxane B₂ (a stable metabolite of thromboxane A₂) and other metabolites have utility in the measurement of platelet activation and for monitoring aspirin therapy.²^,³^,⁴ Modern assays are ELISA based and are normally performed on fresh serum samples that have been generated ex vivo under standard conditions of incubation (e.g., 1 hour at 37°C). The assays provide sensitive, specific, and relatively simple measurements of COX-1 inhibition, and samples can be stored and batched for analysis. Samples from aspirin-treated patients are unable to generate thromboxane. It is recommended that for monitoring aspirin it is important to measure the consequences of inhibiting the biochemical target of the drug, so these assays provide a simple means for monitoring compliance and effectiveness of aspirin therapy.⁵ More recently, ELISAs of 11-dehydro thromboxane B₂ in stabilized and stored urine samples have also become popular and commercialized (AspirinWorks) and provide a simple noninvasive measurement of thromboxane generation that seems to correlate with aspirin efficacy.⁴^,⁵^,⁶ The latter assays have the disadvantage that they are also more nonspecific to the platelet and can measure other sources of thromboxane generation.⁷

Another biochemical marker, the measurement of the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) by flow cytometry, is discussed in section II.C.5 below.

Soluble Markers

α-Granular Proteins

Providing there is a sufficient stimulus platelets secrete the contents of their granules into the plasma. Platelet α-granules contain a number of growth factors, clotting factors, and adhesion molecules, thus providing a way to release high concentrations of important mediators of inflammation, chemotaxis,
and hemostasis within the local environment of the growing platelet thrombus. As some of these markers are also soluble and specific to the megakaryocyte-platelet lineage, their measurement provides a way to systemically and specifically measure the degree of platelet activation occurring in various disease states. Platelet-derived α-granular markers are now usually measured by ELISAs that have superseded radioimmunoassays. Of these molecules, the most commonly and traditionally measured markers of platelet activation are the α-granular soluble constituents, β-thromboglobulin (β-TG),⁸^,⁹ and platelet factor 4 (PF4).⁸^,⁹ More recently, some α-granular membrane proteins have been found to be expressed on the surface of activated platelets where they are rapidly cleaved by proteases and are shed into the plasma. These include soluble P-selectin¹⁰^,¹¹ and soluble CD40 ligand (sCD40L)¹² (see the platelet “sheddome” section below). However, as a result of the obligatory plasma separation procedures, all of these assays are vulnerable to artifactual in vitro platelet activation.⁸^,⁹

Table 67.1 Summary of laboratory markers of platelet activation

Biochemical markers

• Thromboxane (serum, urine)

• VASP

Soluble markers

• α-Granule releasates

• Dense granule releasates

• Platelet “sheddome”

Changes in platelet surface glycoproteins

Platelet surface exposure of granule membrane proteins

Platelet-platelet aggregates

Leukocyte-platelet aggregates

Platelet surface PS

Platelet-derived microparticles

Dense Granular Proteins

Platelet-dense granules contain a unique pool of molecules (e.g., serotonin [5-HT], nucleotides, polyphosphate [Poly-P]) that can also provide a sensitive measurement of platelet activation. Lumiaggregometry can be used to measure the ex vivo release of adenosine triphosphate (ATP) from dense granules during the aggregation response to an exogenous agonist.¹³ Levels of platelet-released ATP and ADP can also be measured by luminometry within standardized platelet-rich plasma preparations that have been activated and lysed.¹⁴ These measurements are useful for the diagnosis of storage and release disorders. Circulating serotonin is actively taken up by platelets and packaged into the dense granules. Some in vitro platelet activation assays utilize the uptake of radio-labeled serotonin and measure its release after stimulation with different agonists. Dense granular Poly-P has recently been shown to be an endogenous activator of factor XII, a component of the intrinsic clotting cascade and proinflammatory pathways.¹⁵^,¹⁶ However, as none of these substances are exclusively synthesized and stored within platelets, their measurement within ex vivo blood samples is not as common as with some of the more specific platelet α-granular-derived or shed glycoproteins.

The Platelet “Sheddome”

It has become increasingly apparent that proteolysis of a large number of different platelet surface and granular membrane proteins during or after platelet activation results in the generation of a growing list of potentially useful plasma markers of platelet activation. These protein fragments have been given the collective term the platelet “sheddome” and are released from the platelet surface by proteinases such as ADAM17 or ADAM10.¹⁷ Shedding tends to be slower than platelet activation and is a selective process rather than a wholesale loss of all surface glycoproteins. Among those proteins shed are glycoprotein (GP) Ib (glycocalicin), GPVI, GPV, P-selectin, endothelial cell-specific adhesion molecule, junctional adhesion molecule-A, semaphorin 4D, and CD40L.¹⁷ Many of the shed protein fragments may play roles in platelet and vascular biology, although the full implications of this shedding process are still largely unknown. Nevertheless, measurement of components of the sheddome may prove to be useful as sensitive and specific biomarkers of platelet activation in vivo. For example, soluble GPVI is an excellent candidate for a specific biomarker of platelet activation as it is found in the plasma at relatively low levels in healthy subjects but is elevated in patients with thrombotic risk, heparin-induced thrombocytopenia, and immune thrombocytopenic purpura.¹⁸^,¹⁹ Exposure of platelets in vitro to high shear stress also results in increased soluble GPVI levels. Further work is required to determine the clinical potential of this unique marker. Platelet surface GPV is also cleaved by thrombin,²⁰^,²¹^,²² releasing a soluble form of GPV (sGPV) into plasma, which may serve as another biomarker of platelet activation.²³ A number of the shed glycoproteins (e.g., CD40L and P-selectin) are also present in α-granule membranes and are only detectable on the surface of activated platelets, where they can be cleaved and shed by the action of proteases. Release of sCD40L from the platelet surface is the predominant source of sCD40L.²⁴^,²⁵ sCD40L is thought to be prothrombotic by stabilizing α_IIbβ₃-dependent arterial thrombosis²⁶ but can also be proinflammatory²⁷^,²⁸ (although not all studies agree on this latter point²⁴). sCD40L levels have been shown to be elevated in patients with acute myocardial infarction and other acute coronary syndromes. P-selectin (CD62P) is also externalized from the α-granules during platelet activation and is gradually cleaved and shed. Increased levels of soluble P-selectin have been found in many diseases and may predict major adverse cardiovascular events.¹¹

An important practical point is that many published patient studies of shed glycoprotein levels were performed using serum. However, blood clotting results from thrombin generation and it is accompanied by platelet activation and the ex vivo release of large amounts of these markers into the serum. Therefore, accurate measurement of in vivo circulating molecules released from platelets requires assays performed using plasma rather than serum.²⁹ Platelets also have to be carefully removed during plasma preparation, without any significant activation; anticoagulants (e.g., citrate, theophylline, adenosine, and dipyridamole [CTAD]) containing some platelet inhibitory agents have
been traditionally used for measurement of PF4 and β-TG to help minimize any additional ex vivo platelet activation. Soluble P-selectin in plasma may also be of endothelial cell origin although it is thought that this is a predominant marker of platelet activation.¹⁰

Flow Cytometric Measurement of Platelet Activation

Introduction

Flow cytometry enables the rapid measurement of characteristics of a large number of individual cells.³⁰ For flow cytometric analysis, cells in suspension are fluorescently labeled, typically with a fluorescently conjugated monoclonal antibody. In the flow cytometer, the suspended cells pass through a focused laser beam at a rate of 1,000 to 10,000 cells per minute. After the laser light activates the fluorophore at the excitation wavelength, detectors process the emitted fluorescence and light scattering properties of each cell.³⁰ The intensity of the emitted light is directly proportional to the antigen density or the characteristics of the cell being measured.

Clinical studies utilizing flow cytometric assays of washed platelets or platelet-rich plasma are, like other assays of platelet function, potentially susceptible to artifactual in vitro platelet activation during obligatory separation procedures. The introduction of whole blood flow cytometry by Shattil et al.³¹ was therefore a major step toward the application of flow cytometry to clinical settings.

A typical schema of sample preparation for whole blood flow cytometry is shown in FIGURE 67.1. The anticoagulant is usually buffered trisodium citrate (105 to 109 mmol/L or 129 mmol/L). Samples are then diluted (typically 1:10) in the presence of antibodies or agonists. The purpose of the dilution is to minimize the formation of platelet aggregates (see below). A minimum of two monoclonal antibodies is used, each conjugated with a different fluorophore. A wide variety of fluorophores are available for antibody conjugation (e.g., phycoerythrin, fluorescein, peridinin chlorophyll protein [PerCP], phycoerythrin-Cy5, phycoerythrin-Texas Red [Red-670], allophycocyanin [APC]). The “test” antibody (the monoclonal antibody recognizing the antigen to be measured) is added at a saturating concentration. The “platelet identifier” monoclonal antibody (e.g., GPIb, integrin α_IIb, or integrin β₃ specific) is added at a near saturating concentration. Physiologic agonists can be used in the assay, including thrombin, thrombin receptor-activating peptides (TRAPs), ADP, collagen, collagen-related peptide (CRP), the complement fraction C5b-9, and thromboxane A₂ analogs. Nonphysiologic agonists include phorbol myristate acetate and the calcium ionophore A23187. Samples are stabilized by fixation, typically with final concentrations of 0.2% to 1% formaldehyde or paraformaldehyde. Antibodies can be added after fixation, provided fixation does not interfere with antibody binding³² (see below). Samples are then analyzed in a flow cytometer. After identification of platelets both by their characteristic light scatter and by the “platelet identifier” antibody, binding of the test monoclonal antibody is determined by analyzing 5,000 to 10,000 individual platelets. During ex vivo activation and labeling, the formation of platelet aggregates is minimized by using very gentle mixing (tapping of the tube) and dilution to minimize collisions between platelets.

FIGURE 67.1 A typical schema of sample preparation for analysis of platelets by whole blood flow cytometry.

For specific protocols of whole blood flow cytometric assay of platelet function, the reader is referred to various references.³³^,³⁴ Laboratory markers of platelet activation include activation-dependent conformational changes in integrin α_IIbβ₃ (aka GPIIb-IIIa, CD41/CD61), exposure of granule membrane proteins, platelet surface binding of secreted platelet proteins, and development of a procoagulant surface (Table 67.2). The two most widely studied types of activation-dependent monoclonal antibodies are those directed against conformational changes in α_IIbβ₃ and those directed against granule membrane proteins.

Changes in Platelet Surface Glycoproteins

The integrin α_IIbß₃ is a receptor for fibrinogen and von Willebrand factor (vWF) that is essential for platelet aggregation.³⁵ Whereas most monoclonal antibodies directed against α_IIbβ₃ bind to resting platelets, the monoclonal antibody PAC1 is directed against the fibrinogen-binding site that is exposed on α_IIbβ₃ by platelet activation (Table 67.1).³⁶ Thus, PAC1 only binds to activated platelets, not to resting platelets. Other α_IIbβ₃-specific activation-dependent monoclonal antibodies are directed against either ligand-induced conformational changes in α_IIbβ₃ (ligand-induced binding sites [LIBS])³⁷ or receptor-induced conformational changes in the bound ligand (fibrinogen) (receptor-induced binding sites [RIBS])³⁸ (Table 67.1). Rather than integrin α_IIbβ₃-specific monoclonal antibodies, fluorescein-conjugated fibrinogen can also be used in flow cytometric assays to detect the activated form of platelet surface α_IIbβ₃,³⁹^,⁴⁰ but the concentration of unlabeled plasma fibrinogen and unlabeled fibrinogen released from platelet α-granules must also be considered in these assays. α_IIbβ₃ is expressed at high copy number on the resting platelet surface, but as this protein is also distributed throughout the open canalicular system (OCS) and α-granular membranes, a significant increase in surface protein
copy density can also be detected on activated platelets. This is normally reflected as an increase in mean fluorescence intensity (MFI) with most fluorescent antibodies against this receptor. Although the vWF receptor GPIb is also expressed at high copy density on resting platelets, changes also occur after activation. As the protein can be internalized into the OCS and/or cleaved to form soluble glycocalicin, significant downregulation in the copy density of this receptor can occur with a decrease in MFI, although the percentage of positive platelets remains 100%.⁴¹^,⁴²

Table 67.2 Activation-dependent monoclonal antibodies, that is, antibodies that bind to activated but not resting platelets

Activation-dependent Surface Change	Prototypic Antibodies	References
*Conformational Changes*
Activation-induced conformational change in integrin α_IIbβ₃, resulting in exposure of the fibrinogen-binding site	PAC1	36
LIBS on integrin α_IIbβ₃	PM 1.1., LIBS1, LIBS6	37,73,219
RIBS on bound fibrinogen	2G5, 9F9, F26	38,117,220
Activation-induced conformational change in integrin α₂β₁	IAC-1	221
*Exposure of Granule Membrane Proteins*
P-selectin (α-granules)	S12, AC1.2	222,223
GMP-33 (α-granules)	RUU-SP 1.77	224,225
CD63 (lysosomes)	CLB-gran/12	226
LAMP-1 (lysosomes)	H5G11	227
LAMP-2 (lysosomes)	H4B4	228
CD40L	TRAP1	92
Lectin-like oxidized LDL receptor (LOX-1)	JTX68	229
*Platelet Surface Binding of Secreted Platelet Proteins*
Thrombospondin	P8, TSP-1	230,231
Multimerin	JS-1	232,233
*Development of a Procoagulant Surface*
Factor V/Va binding	V237	60
Factor X/Xa binding	5224	61
Factor VIII binding	1B3	62
PS exposure	Annexin-V or lactadherin	59,64

Granule Membrane Markers

The most widely studied types of activation-dependent monoclonal antibodies directed against granule membrane proteins are P-selectin (CD62P) specific. P-selectin is a component of the α-granule membrane of resting platelets that is only expressed on the platelet surface membrane after α-granule secretion. Therefore, a P-selectin-specific monoclonal antibody only binds to degranulated platelets, not to resting platelets. The activation-dependent increase in platelet surface P-selectin is not reversible over time in vitro.⁴³^,⁴⁴ However, in vivo circulating degranulated platelets rapidly lose their surface P-selectin, although they continue to circulate and function.⁴⁵^,⁴⁶ Platelet surface P-selectin is therefore not an ideal marker for the detection of circulating degranulated platelets, unless (a) the blood sample is drawn immediately distal to the site of platelet activation, (b) the blood sample is drawn within 5 minutes of the activating stimulus, or (c) there is continuous activation of platelets. The length of time that other activation-dependent markers remain on the platelet surface in vivo has not been definitively determined, but it is becoming clear that some of these are also shed from the surface (see the platelet “sheddome” section above). CD63 is a membrane protein of dense granules and lysosomes and, like P-selectin, it is only expressed upon the surface of activated platelets. However, unlike P-selectin, this glycoprotein is not prone to proteolysis and may provide a more stable marker of platelet activation.

Leukocyte-Platelet Aggregates

P-selectin mediates the initial adhesion of activated platelets to monocytes and neutrophils via the PSGL-1 counterreceptor on the leukocyte surface.⁴⁷ Monocyte-platelet and
neutrophil-platelet aggregates are readily and easily identified by whole blood flow cytometry.³⁴^,⁴⁸ These interactions are important in cardiovascular disease and many other clinical disorders in which platelets are activated (see section on platelet activation in clinical disorders below).⁴⁹^,⁵⁰^,⁵¹

Tracking autologous infused biotinylated platelets in baboons by three-color whole blood flow cytometry directly demonstrated in vivo that (a) platelets degranulated by thrombin very rapidly (within 1 minute) form circulating aggregates with monocytes and neutrophils (FIGURE 67.2, upper panel); (b) the percent of monocytes with adherent infused platelets is greater than the percent of neutrophils with adherent infused platelets (FIGURE 67.2, upper panel); and (c) the in vivo half-life of detectable circulating monocyte-platelet aggregates (˜30 minutes) is longer than both the in vivo half-life of neutrophil-platelet aggregates (˜5 minutes) and the previously reported⁴⁶ rapid loss of surface P-selectin from nonaggregated infused platelets (FIGURE 67.2, upper panel).⁴⁸

These findings suggest that measurement of circulating monocyte-platelet aggregates may be a more sensitive indicator of in vivo platelet activation than either circulating neutrophil-platelet aggregates or circulating P-selectin-positive nonaggregated platelets. Indeed it has been demonstrated that after percutaneous coronary intervention (PCI), there is an increased number of circulating monocyte-platelet (and, to a lesser extent, neutrophil-platelet) aggregates, but not P-selectin-positive platelets, in peripheral blood.⁴⁸ Similarly in patients presenting to an emergency department with chest pain, those with acute myocardial infarction had more circulating monocyte-platelet aggregates than patients without acute myocardial infarction and normal controls. Furthermore, circulating P-selectin-positive platelets were not increased in patients with chest pain with or without acute myocardial infarction.⁴⁸

FIGURE 67.2 In vivo tracking of platelets. Baboons were infused with autologous, biotinylated platelets that were (upper panel) or were not (lower panel) thrombin-activated preinfusion. Surface P-selectin on the infused platelets and participation of the infused platelets in circulating monocyte-platelet and neutrophil-platelet aggregates was determined by three-color whole blood flow cytometric analysis of peripheral blood samples drawn at the indicated time points. The “0” time point refers to blood samples taken immediately preinfusion. Platelet surface P-selectin is expressed as mean fluorescence intensity (MFI), as a percentage of the fluorescence of a preinfusion maximally activated (10 U/mL) thrombin control sample. Monocyte-platelet and neutrophil-platelet aggregates are expressed as the percent of all monocytes and neutrophils with adherent infused platelets. Data are mean ± SEM. (Reproduced from Michelson AD, Barnard MR, Krueger LA, et al. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation 2001;104:1533-1537, with permission.)

Taken together, these studies indicate that circulating monocyte platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin.

Vasodilator-Stimulated Phosphoprotein

Measurement of VASP phosphorylation by flow cytometry can be performed by using a specific monoclonal antibody for VASP and fixed and permeabilized platelets in whole blood.⁵² VASP is an intracellular platelet protein which is not phosphorylated under basal conditions. VASP phosphorylation is regulated by cAMP, and the assay is based upon stimulation/activation of ex vivo blood samples with prostaglandin E₁ (PGE₁) in the presence
and absence of ADP. PGE₁ and ADP (acting via P2Y₁₂) have opposite effects on VASP with either promotion or inhibition of phosphorylation respectively. Therefore the nonphosphorylated VASP correlates with the activity of P2Y₁₂. Interference with the activity of this receptor by the active metabolites of thienopyridines (e.g., clopidogrel or prasugrel) or direct antagonists (e.g., ticagrelor or cangrelor) results in persistent VASP phosphorylation induced by PGE₁ even when the platelets are stimulated with ADP. By comparing the level of VASP phosphorylation in whole blood samples stimulated with either PGE₁ or a combination of PGE₁ and ADP, it is possible to derive a platelet reactivity index (%).⁵³ The assay provides the most direct and specific measurement of the activity of P2Y₁₂ and can be used to monitor the efficacy of antiplatelet drugs that target this receptor.⁵⁴^,⁵⁵ Accordingly, the VASP phosphorylation assay has been used to monitor the effects of clopidogrel in humans, and an impaired restoration of VASP phosphorylation by clopidogrel has been shown to be associated with subacute stent thrombosis following PCI.⁵⁴^,⁵⁵ Further work is required to determine whether this indicates that a high level of receptor blockade is required to prevent stent thrombosis, particularly as the assay has been reported to be insensitive to low levels of P2Y₁₂ inhibition.

Phosphatidylserine

The distribution of phospholipids in the plasma membrane of resting platelets is asymmetric with the negatively charged phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) sequestered in the inner leaflet. The sustained increases in cytosolic calcium elevation that accompanies platelet activation by strong agonists result in the activation of scramblase,⁵⁶ and PS then provides a surface upon which the tenase and prothrombinase complexes can assemble, resulting in a burst of thrombin generation. It is possible to measure the extent of PS exposure by flow cytometry either ex vivo or in vitro after activation by using probes that bind to PS. Annexin-V is a high-affinity calcium-dependent probe for detecting PS and has been extensively used in platelet studies.⁵⁷ Recently, the calcium-independent probe lactadherin has also been utilized and shown to be more sensitive at detecting low levels of PS exposure.⁵⁸^,⁵⁹ Other probes based upon various clotting factors including monoclonal antibodies against factor V or factor VIII or directly labeled proteins have also been used.⁶⁰^,⁶¹^,⁶²

Platelet-Derived Microparticles

The sustained cytosolic calcium elevation that results in PS exposure also causes membrane blebbing and the release of microparticles, some of which are PS positive.⁶³ As determined by flow cytometry, in vitro activation of platelets by agonists such as C5b-9, collagen/thrombin, CRP, and the calcium ionophore A23187 in the presence of extracellular calcium ions results in the formation and release of platelet-derived microparticles. The microparticles are between 100 nm and <1 µm in size as determined by low forward-angle light scatter and binding of a platelet-specific monoclonal antibody and may be procoagulant as determined by binding of monoclonal antibodies to activated factors V or VIII or direct detection using the high-affinity probes annexin-V or lactadherin.⁶⁰^,⁶²^,⁶⁴ Procoagulant platelet-derived microparticles play an important role in the assembly of the “tenase” and “prothrombinase” components of the coagulation system in vivo as exemplified by the very rare inherited platelet disorder “Scott syndrome” where the patients present with a bleeding diathesis and cannot expose PS on cell surfaces or generate microparticles.⁶⁵ There are many methods available for measuring microparticles including ELISA, thrombin generation, and procoagulant assays but flow cytometry is regarded as the method of choice. Many flow cytometric methods for the direct detection of procoagulant platelet-derived microparticles in whole blood have been reported.⁶⁶^,⁶⁷^,⁶⁸^,⁶⁹ However, many flow cytometers are limited in their ability to detect particles below approximately 300 to 500 nm. The use of standardization beads (e.g., Megamix, Biocytex) facilitates optimal resolution of labeled microparticles from electronic noise and improves both within- and between-laboratory standardization.⁶⁸ Platelets can also release exosomes that are true nanoparticles formed within the α-granules from multivesicular bodies.⁷⁰ Platelet and other cellular derived microparticles are elevated in plasma in a wide range of disease including many thrombotic disorders where platelets are activated and thus may be novel disease biomarkers.⁷¹

Methodologic Issues

Preanalytical Considerations: Blood Drawing, Sample Handling, and Processing

It is imperative that any ex vivo artifacts are prevented during the processing and preparation of samples for the measurement of platelet activation markers. Some markers are sensitive to adrenergic stimulation and exercise, so subjects are often rested for at least 10 minutes prior to venipuncture. Platelet activation and aggregate formation can be minimized in the preparation of platelets for whole blood flow cytometry by a combination of the following methods: (a) preparing reagents in advance and avoiding delays in procedure; (b) using a light tourniquet and a needle not narrower than 21 gauge to collect blood; (c) smooth, easy flow from the blood draw; (d) discarding the first few milliliters of blood; (e) using polypropylene (or siliconized glass) tubes or syringes; (f) immediate mixing with the anticoagulant; (g) no washing, centrifugation, gel filtration, vortexing, or stirring steps; (h) reducing the platelet count by dilution of the samples; (i) if thrombin is the agonist, including the synthetic tetrapeptide Gly-Pro-Arg-Pro (GPRP) in the assay (see below); (j) mixing gently after addition of agonist, then incubating undisturbed; (k) fixation (see use of fixatives section below). RGD-containing peptides have also been used to prevent platelet aggregates,⁷² but these peptides may interfere with the binding of detecting antibodies such as PAC1, and they can result in exposure of LIBS.³⁷^,⁷³

Each sample should be monitored for evidence of platelet aggregation (“smearing” of the platelets into the upper right quadrant of the log side [orthogonal] light scatter vs. log forward light scatter histogram). After discarding the first few milliliters, blood should be collected into buffered 3.2% (105 to 109 mmol/L) trisodium citrate Vacutainer tubes (Becton Dickinson, Rutherford, NJ), which does not result in significant platelet activation.⁷⁴ However, each laboratory should determine whether their method of collection, including the drawing of samples through PCI and other catheters, results in artifactual in vitro platelet activation, as determined by the binding of activation-dependent monoclonal antibodies. To measure platelet activation markers, blood should be kept at room temperature and rapidly processed as ex vivo changes in various markers can occur rather quickly (e.g., 10 minutes).

Although the weak calcium chelator sodium citrate is the most commonly used anticoagulant, other anticoagulants have
been successfully used, for example, corn trypsin inhibitor (an inhibitor of activated factor XII).⁷⁵ The strong calcium chelator ethylenediaminetetraacetic acid (EDTA) should be avoided, because it can dissociate α_IIbβ₃. Nonchelating anticoagulants like PPACK (a direct thrombin inhibitor) or hirudin may be preferable for the monitoring of GPIIb-IIIa antagonist therapy.⁷⁶ Citrate has also recently been shown to interfere with in vitro but not ex vivo measurement of P2Y₁₂ inhibition by low concentrations of cangrelor and the prasugrel metabolite.⁷⁷ Another alternative anticoagulant available is EDTA-CTAD.⁷⁸ Platelet count, mean platelet volume (MPV), number of platelet clumps, mean platelet component, numbers of P-selectin-positive platelets, and platelet-leukocyte aggregates have all been shown to be stable for 360 minutes in blood samples kept at 4°C.

Only gold members can continue reading. Log In or Register to continue