Section 1 Platelet Function

**3**

**Chapter 1**

**Abstract**

Platelet Imaging

analysis by electron microscopy.

barriers that should to be addressed.

**1. Introduction**

fluorescence microscopy, intravital microscopy

*Zachary A. Matthay and Lucy Zumwinkle Kornblith*

The knowledge gained through imaging platelets has formed the backbone of our understanding of their biology in health and disease. Early investigators relied on conventional light microscopy with limited resolution and were primarily able to identify the presence and basic morphology of platelets. The advent of high resolution technologies, in particular, electron microscopy, accelerated our understanding of the dynamics of platelet ultrastructure dramatically. Further refinements and improvements in our ability to localize and reliably identify platelet structures have included the use of immune-labeling techniques, correlativefluorescence light and electron microscopy, and super-resolution microscopies. More recently, the expanded development and application of intravital microscopy in animal models has enhanced our knowledge of platelet functions and thrombus formation *in vivo*, as these experimental systems most closely replicate native biological environments. Emerging improvements in our ability to characterize platelets at the ultrastructural and organelle levels include the use of platelet cryogenic electron tomography with quantitative, unbiased imaging analysis, and the ability to genetically label platelet features with electron dense markers for

**Keywords:** platelets, platelet disorders, electron microscopy, confocal microscopy,

Platelets are small, anucleate cells with central roles in hemostasis, endothelial regulation, inflammation, and immune responses [1–5]. Critical to these biologic processes, platelets undergo several dynamic morphologic changes in overall shape and size. Furthermore, at the organelle level they alter and differentially release their cellular contents in signal dependent manners [6–10]. Detailed knowledge of these changes is essential for understanding platelet biology, including in the contexts of hemostasis and thrombosis, cellular and pathogen interactions, and intracellular signaling mechanisms [9, 11–14]. Within this chapter, we will review the methods and applications of multiple modalities of platelet imaging including light transmission, super-resolution, electron, and intravital microscopy. Advances in our ability to image platelets have enabled a more complete understanding of platelet biology in health and disease, and these techniques remain powerful clinical and research tools [15–19]. We will also detail the challenges of these imaging methods specific to platelets, and future

## **Chapter 1** Platelet Imaging

*Zachary A. Matthay and Lucy Zumwinkle Kornblith*

## **Abstract**

The knowledge gained through imaging platelets has formed the backbone of our understanding of their biology in health and disease. Early investigators relied on conventional light microscopy with limited resolution and were primarily able to identify the presence and basic morphology of platelets. The advent of high resolution technologies, in particular, electron microscopy, accelerated our understanding of the dynamics of platelet ultrastructure dramatically. Further refinements and improvements in our ability to localize and reliably identify platelet structures have included the use of immune-labeling techniques, correlativefluorescence light and electron microscopy, and super-resolution microscopies. More recently, the expanded development and application of intravital microscopy in animal models has enhanced our knowledge of platelet functions and thrombus formation *in vivo*, as these experimental systems most closely replicate native biological environments. Emerging improvements in our ability to characterize platelets at the ultrastructural and organelle levels include the use of platelet cryogenic electron tomography with quantitative, unbiased imaging analysis, and the ability to genetically label platelet features with electron dense markers for analysis by electron microscopy.

**Keywords:** platelets, platelet disorders, electron microscopy, confocal microscopy, fluorescence microscopy, intravital microscopy

## **1. Introduction**

Platelets are small, anucleate cells with central roles in hemostasis, endothelial regulation, inflammation, and immune responses [1–5]. Critical to these biologic processes, platelets undergo several dynamic morphologic changes in overall shape and size. Furthermore, at the organelle level they alter and differentially release their cellular contents in signal dependent manners [6–10]. Detailed knowledge of these changes is essential for understanding platelet biology, including in the contexts of hemostasis and thrombosis, cellular and pathogen interactions, and intracellular signaling mechanisms [9, 11–14]. Within this chapter, we will review the methods and applications of multiple modalities of platelet imaging including light transmission, super-resolution, electron, and intravital microscopy. Advances in our ability to image platelets have enabled a more complete understanding of platelet biology in health and disease, and these techniques remain powerful clinical and research tools [15–19]. We will also detail the challenges of these imaging methods specific to platelets, and future barriers that should to be addressed.

## **2. Light and fluorescence microscopy**

## **2.1 Overview**

Owing to their small size, platelets were not discovered until long after red blood cells and leukocytes had been visualized with traditional microscopy. With the development of compound and achromatic microscopes, they were finally observed by several scientists in the nineteenth century, including the Italian scientist Giulio Bizzozero, who began to identify their roles in hemostasis and thrombosis [14, 20]. In their resting state, platelets are 2–3 μm in diameter and discoid in shape, appearing as small blue circular structures on traditional light microscopy with Giemsa staining (**Figure 1**). Despite the resolution limitations of light microscopy, many important advances have been made in our understanding of platelet structure and function with this technique. In fact, light microscopy remains an important diagnostic and research tool today [21, 22].

## **2.2 Diagnosis of platelet disorders with light and fluorescence microscopy**

Many heritable platelet disorders can be diagnosed using light microscopy of blood smears when used in conjunction with immunofluorescent staining. Typically, light microscopy is initially used to identify abnormalities in platelet shape and size, narrowing the differential diagnosis, which is then followed by specific staining or flow cytometry to make a final diagnosis (**Figure 2**) [21]. This may be diagnostic in approximately 25–35% of cases of suspected inherited thrombocytopenia, and can obviate the need for further expensive genetic testing or complex imaging modalities such as electron microscopy [21, 22].

## **2.3 Platelet spreading assays**

Differential interference contrast microscopy is a specialized form of light microscopy used to characterize platelet spreading and adhesion to proteins on transparent surfaces [24]. For example, the role of actin proteins in the platelet

#### **Figure 1.**

*Standard light microscopy of platelets. Image from a light microscope (500×) from a Giemsa-stained peripheral blood smear showing platelets (blue dots) surrounded by red blood cells (pink circular structures). Image and caption by Dr. Graham Beards, reproduced under Creative Commons GNU Free Documentation 1.0 Generic license (https://creativecommons.org/licenses/by-sa/1.0/deed.en).*

**5**

**Figure 3.**

*org/licenses/by/2.0).*

*Platelet Imaging*

**Figure 2.**

*Sandrock et al. [23].*

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

**2.4 Imaging platelets under flow**

responses to collagen or fibrinogen can be studied (**Figure 3**) [25]. This form of microscopy enhances contrast in unstained platelet samples thereby allowing adequate visualization of shape change [24]. Platelet spreading experiments have led to several important advances in platelet biology such as the identification of sex differences in platelet adhesion [26], discovery of novel platelet structures such as actin nodules [27], and delineation of platelet-extracellular matrix interaction [25, 28].

*Light microscopy of peripheral blood smear. Performed with Giemsa staining, showing a giant platelet characteristic of Bernard-Soulier syndrome. Diagnosis can then be confirmed by either immune-fluorescent microscopy or flow cytometry demonstrating defects in glycoprotein Ib. Image reproduced with permission from* 

In order to study platelet behavior in conditions that more closely replicate human vascular biology, techniques that incorporate the imaging of platelets under flow conditions have been developed [8, 29–31]. These include *in vitro* methods such as "flow chambers" and *in vivo* intravital microscopy (detailed in Section 5). Flow chambers include a wide range of home-made and commercially available

*and HS1 (actin binding protein) −/− mice were added to cover slips coated with collagen (100 μg/ml), collagen related peptide (10 μg/ml) or fibrinogen (100 μg/ml) ± thrombin (1 U/ml) and allowed to settle for 45 or 90 min at 37°C. Spread platelets were fixed in formalin and imaged using differential interference contrast microscopy. Representative images of platelets at 45 min are shown. Image and caption reproduced with permission from Thomas et al. [25] under Creative Commons Attribution License (http://creativecommons.*

 *platelets/ml) from wild type* 

*Analysis of platelet spreading and actin organization. Washed platelets (2 × 107*

#### **Figure 2.**

*Platelets*

**2.1 Overview**

**2. Light and fluorescence microscopy**

diagnostic and research tool today [21, 22].

**2.3 Platelet spreading assays**

imaging modalities such as electron microscopy [21, 22].

*license (https://creativecommons.org/licenses/by-sa/1.0/deed.en).*

Owing to their small size, platelets were not discovered until long after red blood

cells and leukocytes had been visualized with traditional microscopy. With the development of compound and achromatic microscopes, they were finally observed by several scientists in the nineteenth century, including the Italian scientist Giulio Bizzozero, who began to identify their roles in hemostasis and thrombosis [14, 20]. In their resting state, platelets are 2–3 μm in diameter and discoid in shape, appearing as small blue circular structures on traditional light microscopy with Giemsa staining (**Figure 1**). Despite the resolution limitations of light microscopy, many important advances have been made in our understanding of platelet structure and function with this technique. In fact, light microscopy remains an important

**2.2 Diagnosis of platelet disorders with light and fluorescence microscopy**

Many heritable platelet disorders can be diagnosed using light microscopy of blood smears when used in conjunction with immunofluorescent staining. Typically, light microscopy is initially used to identify abnormalities in platelet shape and size, narrowing the differential diagnosis, which is then followed by specific staining or flow cytometry to make a final diagnosis (**Figure 2**) [21]. This may be diagnostic in approximately 25–35% of cases of suspected inherited thrombocytopenia, and can obviate the need for further expensive genetic testing or complex

Differential interference contrast microscopy is a specialized form of light microscopy used to characterize platelet spreading and adhesion to proteins on transparent surfaces [24]. For example, the role of actin proteins in the platelet

*Standard light microscopy of platelets. Image from a light microscope (500×) from a Giemsa-stained peripheral blood smear showing platelets (blue dots) surrounded by red blood cells (pink circular structures). Image and caption by Dr. Graham Beards, reproduced under Creative Commons GNU Free Documentation 1.0 Generic* 

**4**

**Figure 1.**

*Light microscopy of peripheral blood smear. Performed with Giemsa staining, showing a giant platelet characteristic of Bernard-Soulier syndrome. Diagnosis can then be confirmed by either immune-fluorescent microscopy or flow cytometry demonstrating defects in glycoprotein Ib. Image reproduced with permission from Sandrock et al. [23].*

responses to collagen or fibrinogen can be studied (**Figure 3**) [25]. This form of microscopy enhances contrast in unstained platelet samples thereby allowing adequate visualization of shape change [24]. Platelet spreading experiments have led to several important advances in platelet biology such as the identification of sex differences in platelet adhesion [26], discovery of novel platelet structures such as actin nodules [27], and delineation of platelet-extracellular matrix interaction [25, 28].

## **2.4 Imaging platelets under flow**

In order to study platelet behavior in conditions that more closely replicate human vascular biology, techniques that incorporate the imaging of platelets under flow conditions have been developed [8, 29–31]. These include *in vitro* methods such as "flow chambers" and *in vivo* intravital microscopy (detailed in Section 5). Flow chambers include a wide range of home-made and commercially available

#### **Figure 3.**

*Analysis of platelet spreading and actin organization. Washed platelets (2 × 107 platelets/ml) from wild type and HS1 (actin binding protein) −/− mice were added to cover slips coated with collagen (100 μg/ml), collagen related peptide (10 μg/ml) or fibrinogen (100 μg/ml) ± thrombin (1 U/ml) and allowed to settle for 45 or 90 min at 37°C. Spread platelets were fixed in formalin and imaged using differential interference contrast microscopy. Representative images of platelets at 45 min are shown. Image and caption reproduced with permission from Thomas et al. [25] under Creative Commons Attribution License (http://creativecommons. org/licenses/by/2.0).*

devices such as parallel-plate flow chambers or biochips with micro channels through which fluid is passed at arterial or venous shear stresses [32, 33]. Confocal and fluorescence microscopy is then used to visualize platelet adhesion, aggregation and thrombus formation [30, 33]. Investigators can create a range of experimental conditions to study platelet behavior which has led to many advances in our understanding of platelet biology, thrombus formation, and responses to antiplatelet therapies [29, 32]. Most recently, for example, Tunstromer et al. used platelet staining and time-lapse microscopy to track the movements of individual platelets during thrombus formation using a flow chamber [29]. This model was applied to quantitatively study the effects of platelet inhibitors on platelet contraction, revealing differential impacts on platelet subpopulations [29].

## **3. Super-resolution microscopy**

## **3.1 Overview**

Standard light microscopy offers a maximum resolution of about 200–300 nm due to the limits on resolution to about half of the wavelength of visible light [34]. This allows platelets to be identified, but it is difficult to distinguish and examine intracellular structures. However, recent advances in fluorescence light microscopy with the development of super-resolution microscopy techniques have improved upon conventional light microscopy resolution by 2–10 fold, to the 10–200 nm range, and the pioneers of these advances were awarded Nobel prize in chemistry in 2014 [34, 35]. These novel techniques all utilize fluorescent labeling but broadly apply three different imaging techniques (wide-field microscopy, total internal reflection fluorescence, or confocal microscopy) to overcome the resolution limit of light microscopy, with resolutions at the 10–200 nm range [36, 37]. Further, they can be coupled to automated image analysis to systematically evaluate platelet granules and reveal three dimensional structural details [34, 37].

## **3.2 Localization microscopy or pointillist imaging by single-molecule localization (SMLM)**

Localization microscopy or pointillist imaging by single-molecule localization (SMLM) entails using many cycles of detecting and localizing single fluorescent labels within the cell achieving up to 10 nm resolution. By repeating the fluorescent imaging cycles thousands of times in conjunction with an on/off mechanism for fluorescence, the positions of molecules are precisely mapped out [37]. However, this process is quite slow given the need for several cycles of images to be taken. Sub-variants include photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) [34].

### **3.3 Target based inhibition of fluorescence emission by stimulated emission**

Target based inhibition of fluorescence emission by stimulated emission uses an excitation and a depletion laser beam to manipulate fluorescent signals to further improve resolution, relying on diffraction properties of light from the microscope's focal point [37, 38]. Some examples of this include stimulated emission depletion (STED), reversible saturable optical fluorescence transitions (RESOLFT), and conical diffraction [34]. This method requires a narrow field and imaging an entire sample is a lengthy process, though the resolution is excellent at up to 20 nm [34].

**7**

*Platelet Imaging*

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

**3.4 Structured illumination microscopy**

illumination microscopy [39].

**4.1 Overview of electron microscopy**

discuss their respective advantages and drawbacks.

**4.2 Scanning and transmission electron microscopy**

**cryogenic**

Structured illumination microscopy only improves resolution by approximately 2 fold (up to 120 nm) compared to light microscopy, but this is a more efficient, faster method [35]. It employs mathematical processing of a series of illumination and laser interference patterns to improve resolution [37]. As a proof of concept, Westmoreland and colleagues used structured illumination microscopy to discriminate between three patients with Hermansky-Pudlak Syndrome (a platelet storage defect whereby dense granules are absent) and healthy controls [35]. This work highlights some of the inherent limitations of super-resolution microscopy. Because it relies upon fluorescent labeling of structures of interest, there are risks of mislabeling and therefore misidentification of structures. For example, in their paper, the authors noted that in order to stain dense granules fluorescently, a CD63 marker was used, however, off target effects such as identification of lysosomes were seen as these are also known to be CD63 positive. The description of platelet actin nodules required for platelet-platelet interactions and their potential relevance to Wiskot-Aldrich syndrome is another advance that was made using structured

In summary, super-resolution microscopy is a recent and notable advance over traditional light microscopy techniques in the identification and visualization of platelet ultrastructural features. Despite some limitations, it does offer the advantage, particularly for structured illumination microscopy, of being relatively less time consuming and intensive than other advanced imaging techniques (such as electron microscopy), and as such may ultimately become more widely available.

Electron microscopy (EM) utilizes a beam of accelerated electrons as the source of illumination, and because the wavelength of an electron is up to 100,000 times shorter than that of visible light, this provides resolution at the nanometer level with excellent visualization of platelet ultrastructural details (see **Figure 4**). EM was first developed in the 1930s, and much of our detailed knowledge of platelets has been gleaned through application of EM, including through transmission, scanning, and cryogenic EM [40, 41]. Substantial early contributions were made by James White who analyzed platelet ultrastructure in health and disease states using EM [13, 14, 16, 42–48], and by Marcel Bessis, who studied both megakaryocyte and platelet ultrastructure in addition to other blood cell morphologies [49, 50]. In this section, we will explore the applications of EM in clinical and research settings and

Scanning (SEM) and transmission electron microscopy (TEM) can magnify up to a resolution of 0.2 nm [15]. In SEM, detailed topographical images are obtained of the platelet surface. However, to examine the contents of platelets, TEM is used which sends a beam of electrons through ultrathin sections, providing excellent internal details. EM relies on differences in electron density to differentiate individual structures; however, some structures may have similar electron densities. For example, the identification and study of platelet lysosomes with EM requires

**4. Electron microscopy of platelets: transmission, scanning, and** 

devices such as parallel-plate flow chambers or biochips with micro channels through which fluid is passed at arterial or venous shear stresses [32, 33]. Confocal and fluorescence microscopy is then used to visualize platelet adhesion, aggregation and thrombus formation [30, 33]. Investigators can create a range of experimental conditions to study platelet behavior which has led to many advances in our understanding of platelet biology, thrombus formation, and responses to antiplatelet therapies [29, 32]. Most recently, for example, Tunstromer et al. used platelet staining and time-lapse microscopy to track the movements of individual platelets during thrombus formation using a flow chamber [29]. This model was applied to quantitatively study the effects of platelet inhibitors on platelet contraction, reveal-

Standard light microscopy offers a maximum resolution of about 200–300 nm due to the limits on resolution to about half of the wavelength of visible light [34]. This allows platelets to be identified, but it is difficult to distinguish and examine intracellular structures. However, recent advances in fluorescence light microscopy with the development of super-resolution microscopy techniques have improved upon conventional light microscopy resolution by 2–10 fold, to the 10–200 nm range, and the pioneers of these advances were awarded Nobel prize in chemistry in 2014 [34, 35]. These novel techniques all utilize fluorescent labeling but broadly apply three different imaging techniques (wide-field microscopy, total internal reflection fluorescence, or confocal microscopy) to overcome the resolution limit of light microscopy, with resolutions at the 10–200 nm range [36, 37]. Further, they can be coupled to automated image analysis to systematically evaluate platelet

ing differential impacts on platelet subpopulations [29].

granules and reveal three dimensional structural details [34, 37].

tic optical reconstruction microscopy (STORM) [34].

**3.2 Localization microscopy or pointillist imaging by single-molecule** 

Localization microscopy or pointillist imaging by single-molecule localization (SMLM) entails using many cycles of detecting and localizing single fluorescent labels within the cell achieving up to 10 nm resolution. By repeating the fluorescent imaging cycles thousands of times in conjunction with an on/off mechanism for fluorescence, the positions of molecules are precisely mapped out [37]. However, this process is quite slow given the need for several cycles of images to be taken. Sub-variants include photoactivation localization microscopy (PALM) and stochas-

**3.3 Target based inhibition of fluorescence emission by stimulated emission**

Target based inhibition of fluorescence emission by stimulated emission uses an excitation and a depletion laser beam to manipulate fluorescent signals to further improve resolution, relying on diffraction properties of light from the microscope's focal point [37, 38]. Some examples of this include stimulated emission depletion (STED), reversible saturable optical fluorescence transitions (RESOLFT), and conical diffraction [34]. This method requires a narrow field and imaging an entire sample is a lengthy process, though the resolution is excellent at

**3. Super-resolution microscopy**

**localization (SMLM)**

**3.1 Overview**

**6**

up to 20 nm [34].

## **3.4 Structured illumination microscopy**

Structured illumination microscopy only improves resolution by approximately 2 fold (up to 120 nm) compared to light microscopy, but this is a more efficient, faster method [35]. It employs mathematical processing of a series of illumination and laser interference patterns to improve resolution [37]. As a proof of concept, Westmoreland and colleagues used structured illumination microscopy to discriminate between three patients with Hermansky-Pudlak Syndrome (a platelet storage defect whereby dense granules are absent) and healthy controls [35]. This work highlights some of the inherent limitations of super-resolution microscopy. Because it relies upon fluorescent labeling of structures of interest, there are risks of mislabeling and therefore misidentification of structures. For example, in their paper, the authors noted that in order to stain dense granules fluorescently, a CD63 marker was used, however, off target effects such as identification of lysosomes were seen as these are also known to be CD63 positive. The description of platelet actin nodules required for platelet-platelet interactions and their potential relevance to Wiskot-Aldrich syndrome is another advance that was made using structured illumination microscopy [39].

In summary, super-resolution microscopy is a recent and notable advance over traditional light microscopy techniques in the identification and visualization of platelet ultrastructural features. Despite some limitations, it does offer the advantage, particularly for structured illumination microscopy, of being relatively less time consuming and intensive than other advanced imaging techniques (such as electron microscopy), and as such may ultimately become more widely available.

## **4. Electron microscopy of platelets: transmission, scanning, and cryogenic**

#### **4.1 Overview of electron microscopy**

Electron microscopy (EM) utilizes a beam of accelerated electrons as the source of illumination, and because the wavelength of an electron is up to 100,000 times shorter than that of visible light, this provides resolution at the nanometer level with excellent visualization of platelet ultrastructural details (see **Figure 4**). EM was first developed in the 1930s, and much of our detailed knowledge of platelets has been gleaned through application of EM, including through transmission, scanning, and cryogenic EM [40, 41]. Substantial early contributions were made by James White who analyzed platelet ultrastructure in health and disease states using EM [13, 14, 16, 42–48], and by Marcel Bessis, who studied both megakaryocyte and platelet ultrastructure in addition to other blood cell morphologies [49, 50]. In this section, we will explore the applications of EM in clinical and research settings and discuss their respective advantages and drawbacks.

#### **4.2 Scanning and transmission electron microscopy**

Scanning (SEM) and transmission electron microscopy (TEM) can magnify up to a resolution of 0.2 nm [15]. In SEM, detailed topographical images are obtained of the platelet surface. However, to examine the contents of platelets, TEM is used which sends a beam of electrons through ultrathin sections, providing excellent internal details. EM relies on differences in electron density to differentiate individual structures; however, some structures may have similar electron densities. For example, the identification and study of platelet lysosomes with EM requires

#### **Figure 4.**

*Resting platelet shape and structural components. (a) Transmission electron microscopy in the equatorial plane of a resting, discoid platelet, with showing the peripheral MTC in the equatorial plane. (b) A scheme of a platelet in the equatorial plane (upper image) and in cross section (image at the bottom).* Abbreviations*: MTC, microtubular coil DTS dense tubular system; Gly, glycogen; α, α-granules; δ, δ-granules or dense bodies; λ, λ-granules or lysosomes; GR, Golgi remnants; MF, microfilaments; Mit, mitochondria; OCS, open canalicular system; P, pores of the OCS; Rib, ribosomes. Figure and caption reproduced from Neumüller et al. [51], under open access Creative Commons 3.0 Licensing (https://creativecommons.org/licenses/by-sa/3.0/deed.en).*

staining with acid phosphatase or by using arylsulfatase reactive granules to differentiate them from alpha granules [52]. EM has also been used to demonstrate how platelets may release organelles encapsulated in vesicles, such as mitochondria, to drive pathologic inflammation [53, 54].

Despite the significant advances SEM and TEM contributed to the understanding of platelet structure and biology, they have important limitations. Both SEM and TEM require extensive processing of the samples. This includes fixation, dehydration, hardening or embedding, as well as sectioning. Given that platelets are prone to activation, degranulation, and microparticle release with even modest environmental stimuli, the concern is that these fixation processes risk significantly altering and activating platelets. The most common fixative used for SEM and TEM of platelets is glutaraldehyde, which preserves morphology best of the agents used. Despite these limitations, SEM and TEM have significantly contributed to our understanding of the intracellular organization of platelet contents with nanometer resolutions. Critically, SEM and TEM can be combined with immune-gold labeling to further localize structures [7]. For example, this labeling approach has been used to delineate the intracellular distribution of vWF [10], and to demonstrate that thrombin but not adenosine diphosphate treated platelets have upregulated GLUT-3 receptors [55].

#### **4.3 Cryogenic-electron microscopy**

Cryogenic electron microscopy (Cryo-EM), developed in the 1980s, is a technique in which EM is applied to samples which are nearly instantaneously cooled to cryogenic temperatures in aqueous solution [56]. This process (typically done by plunging into liquid ethane) occurs so rapidly that water molecules do not rearrange into crystalline form (also known as amorphous or vitreous ice). This preserves cellular structures such as lipid membranes in their near native states [41]. This is a particularly important advantage over TEM and SEM which require extensive fixation as described above because platelets are known to activate with minimal stimuli. Furthermore, samples can then be stored for long periods of time in liquid

**9**

**Figure 5.**

*licenses/by-sa/3.0/deed.en).*

*Platelet Imaging*

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

analysis of platelet ultrastructural features.

granules [57, 58] (**Figure 5**).

nitrogen. Once cryogenically preserved, standard TEM techniques are applied to the samples. Additionally, multiple "tilt-series" or tomograms are obtained, in which images are captured at multiple angles, which can then be built into 3D reconstructions (also called cryogenic electron tomography or cryo-ET), which can detail internal structures such as the open canalicular system, organelles, and

There are several important diagnostic applications of EM for assisting in the diagnosis of a variety of platelet disorders, particularly for granule defects [15]. Further, there have been recent increases in interest in utilizing EM for platelet research, in particular with the advent of cryo-ET paired with quantitative image

The diagnosis of several granule defects is assisted or can even be made using EM. For example, in Gray Platelet Syndrome, which consists of a rare, heterogenous group of inherited platelet disorders, "gray" and enlarged platelets are observed by EM after Wright staining [59, 60]. More specifically, EM is able to reliably demonstrate the absence of alpha granules and presence of empty vacuoles (which are the immature alpha granule precursors), a key diagnostic criterion [15]. Furthermore, cryogenic electron microscopy studies have revealed different alpha granule subtypes, some of which are in fact tubular in nature and lack vWF [7]. Paris Trousseau syndrome is a rare platelet bleeding disorder also present in nearly all patients with Jacobsen syndrome (chromosomal disorder with multiple anomalies), and the diagnosis can be confirmed by detecting the presence of giant alpha granules by EM with immune-gold labeling [16]. Similarly, for Hermansky-Pudlak syndrome,

*Electron tomographic 3D model of the open canalicular system (OCS). The OCS is shown with a pore (P) connecting it with the surrounding milieu. The DTS is indicated by arrowheads. One virtual slice is shown in background.* Abbreviations*: MTC, microtubular coil DTS dense tubular system; α, α-granules; Mit, mitochondria; OCS, open canalicular system; P, pores of the OCS. Figure and caption reproduced from Neumüller et al. [51], under open access Creative Commons 3.0 Licensing (https://creativecommons.org/*

**4.4 Diagnostic and research applications of electron microscopy**

#### *Platelet Imaging DOI: http://dx.doi.org/10.5772/intechopen.91736*

*Platelets*

**Figure 4.**

staining with acid phosphatase or by using arylsulfatase reactive granules to differentiate them from alpha granules [52]. EM has also been used to demonstrate how platelets may release organelles encapsulated in vesicles, such as mitochondria,

*Resting platelet shape and structural components. (a) Transmission electron microscopy in the equatorial plane of a resting, discoid platelet, with showing the peripheral MTC in the equatorial plane. (b) A scheme of a platelet in the equatorial plane (upper image) and in cross section (image at the bottom).* Abbreviations*: MTC, microtubular coil DTS dense tubular system; Gly, glycogen; α, α-granules; δ, δ-granules or dense bodies; λ, λ-granules or lysosomes; GR, Golgi remnants; MF, microfilaments; Mit, mitochondria; OCS, open canalicular system; P, pores of the OCS; Rib, ribosomes. Figure and caption reproduced from Neumüller et al. [51], under open access Creative Commons 3.0 Licensing (https://creativecommons.org/licenses/by-sa/3.0/deed.en).*

Despite the significant advances SEM and TEM contributed to the understanding of platelet structure and biology, they have important limitations. Both SEM and TEM require extensive processing of the samples. This includes fixation, dehydration, hardening or embedding, as well as sectioning. Given that platelets are prone to activation, degranulation, and microparticle release with even modest environmental stimuli, the concern is that these fixation processes risk significantly altering and activating platelets. The most common fixative used for SEM and TEM of platelets is glutaraldehyde, which preserves morphology best of the agents used. Despite these limitations, SEM and TEM have significantly contributed to our understanding of the intracellular organization of platelet contents with nanometer resolutions. Critically, SEM and TEM can be combined with immune-gold labeling to further localize structures [7]. For example, this labeling approach has been used to delineate the intracellular distribution of vWF [10], and to demonstrate that thrombin but not adenosine diphosphate treated platelets have upregulated GLUT-3 receptors [55].

Cryogenic electron microscopy (Cryo-EM), developed in the 1980s, is a technique in which EM is applied to samples which are nearly instantaneously cooled to cryogenic temperatures in aqueous solution [56]. This process (typically done by plunging into liquid ethane) occurs so rapidly that water molecules do not rearrange into crystalline form (also known as amorphous or vitreous ice). This preserves cellular structures such as lipid membranes in their near native states [41]. This is a particularly important advantage over TEM and SEM which require extensive fixation as described above because platelets are known to activate with minimal stimuli. Furthermore, samples can then be stored for long periods of time in liquid

to drive pathologic inflammation [53, 54].

**4.3 Cryogenic-electron microscopy**

**8**

nitrogen. Once cryogenically preserved, standard TEM techniques are applied to the samples. Additionally, multiple "tilt-series" or tomograms are obtained, in which images are captured at multiple angles, which can then be built into 3D reconstructions (also called cryogenic electron tomography or cryo-ET), which can detail internal structures such as the open canalicular system, organelles, and granules [57, 58] (**Figure 5**).

## **4.4 Diagnostic and research applications of electron microscopy**

There are several important diagnostic applications of EM for assisting in the diagnosis of a variety of platelet disorders, particularly for granule defects [15]. Further, there have been recent increases in interest in utilizing EM for platelet research, in particular with the advent of cryo-ET paired with quantitative image analysis of platelet ultrastructural features.

The diagnosis of several granule defects is assisted or can even be made using EM. For example, in Gray Platelet Syndrome, which consists of a rare, heterogenous group of inherited platelet disorders, "gray" and enlarged platelets are observed by EM after Wright staining [59, 60]. More specifically, EM is able to reliably demonstrate the absence of alpha granules and presence of empty vacuoles (which are the immature alpha granule precursors), a key diagnostic criterion [15]. Furthermore, cryogenic electron microscopy studies have revealed different alpha granule subtypes, some of which are in fact tubular in nature and lack vWF [7]. Paris Trousseau syndrome is a rare platelet bleeding disorder also present in nearly all patients with Jacobsen syndrome (chromosomal disorder with multiple anomalies), and the diagnosis can be confirmed by detecting the presence of giant alpha granules by EM with immune-gold labeling [16]. Similarly, for Hermansky-Pudlak syndrome,

#### **Figure 5.**

*Electron tomographic 3D model of the open canalicular system (OCS). The OCS is shown with a pore (P) connecting it with the surrounding milieu. The DTS is indicated by arrowheads. One virtual slice is shown in background.* Abbreviations*: MTC, microtubular coil DTS dense tubular system; α, α-granules; Mit, mitochondria; OCS, open canalicular system; P, pores of the OCS. Figure and caption reproduced from Neumüller et al. [51], under open access Creative Commons 3.0 Licensing (https://creativecommons.org/ licenses/by-sa/3.0/deed.en).*

the absence of dense granules on EM is criteria for diagnosis [17], while Chediak-Higashi syndrome can be confirmed by observing decreased dense bodies, normal alpha granules, and giant cytoplasmic inclusions [46]. While the clinical use of EM is not widely available, it remains an important tool for the diagnosis of rare, but important platelet disorders described above, as well as for several others [15, 46].

In addition to the current clinical applications of platelet EM for the diagnosis of platelet disorders, emerging research using cryo-ET shows promise for expanding our understanding of the role of platelets in a range of disease states including cancer [61], thrombo-inflammatory conditions, and for monitoring the quality of blood banking strategies for platelets [51, 58]. In a recent investigation using cryo-ET of platelets from patients with ovarian cancer, the authors identified important morphological changes in nine parameters between those with and without malignancy, including shortened microtubules, and increases in both size and number of platelet mitochondria [61]. These differences were then incorporated into a prediction model which accurately identified 20 out of 23 patients with ovarian cancer, demonstrating exciting potential for future diagnostic purposes.

Electron microscopy has also been used to characterize the properties of platelet microparticles, patterns of platelet activation, as well as the structure of platelet plug formation in *in vivo* models [18, 58, 62, 63]. For example, Ponomareva et al used both SEM and TEM to study the generation of microparticles from platelets in response to stimulation from adenosine diphosphate, arachidonic acid, and thrombin, and found that many microparticles were in fact smaller (less than 300 nm) than those typically able to be detected by flow cytometry [18]. Furthermore, they found that microparticles isolated from patients with conditions such as disseminated intravascular coagulation, heparin induced thrombocytopenia, or lupus were larger and displayed pathologic properties [18]. Lastly, in an *in vivo* mouse model of vascular injury, Tomaiuolo et al used a combination of fluorescence microscopy and SEM to detail different levels of platelet activation within a forming thrombus, and the effects of antiplatelet agents on the hemostatic plug's architecture [63].

#### **4.5 Current limitations and future directions in electron microscopy**

Despite its clear potential as both a research and clinical tool, EM has important drawbacks. In addition to high costs, it requires specialized equipment and highly trained personnel, and therefore is neither widely available nor easy to automate. Furthermore, from a technical perspective, sample thickness is limited, as resolution dramatically decreases with increasing diameters, though this presents more of a challenge for imaging larger cells than it does for platelets [41]. Further, if an intracellular structure takes on an unexpected shape or localization, it can be very difficult to identify without the aid of immuno-labeling, which is not always feasible. Currently, techniques to label intracellular structures require fluorescent tagging and light microscopy, which can be correlated to electron microscopy images (correlative light and electron microscopy or CLEM). These have been improved upon with the use of super-resolution light microscopies as described above, but still does not reach the resolution of EM (super-resolution correlated light and electron microscopy) [35, 36, 41, 64]. However, emerging techniques using cloneable, genetic, electron dense markers, for direct localization with EM could have important applications if used with EM of platelets [65, 66]. Lastly, expanding the use of EM for diagnostic purposes, not just of rare platelet disorders, but in other conditions which affect platelets such as malignancy may provide important insights into disease processes as well as novel diagnostic approaches [61].

**11**

**5.3 Applications and examples**

*Platelet Imaging*

**5.1 Overview**

**function** *in vivo*

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

**5.2 Choice of site and imaging technique**

**5. Intravital microscopy: imaging thrombus formation and platelet** 

and distinctive platelet activation and signaling patterns [9, 11, 70, 71].

The cremaster muscle and mesenteric veins of mice are the most commonly used imaging windows for intravital microscopy, but other possible sites include carotid artery, brain, skin, bones, or liver [11, 72, 73]. In order for visualization and subsequent quantitative analyses, platelets must be labeled with fluorescent antibodies or genetically modified mouse models which express fluorescent platelet proteins. Both of these approaches provide adequate capacity to visualize platelets *in vivo*, however, at least for some of the genetic approaches in mice, there may be untoward effects on platelet aggregation compared to antibody labeling approaches [74]. While any vascular bed theoretically may be used to study platelet function, the primary limitations include the anatomic challenges of isolating the vessels while minimizing trauma and inflammation, as well as vessel caliber, since it must be sufficiently transparent for light penetration [75]. Pros and cons of each anatomic site are summarized below (**Table 1**). The essential imaging equipment includes a microscope, an illumination light source for fluorescence excitation, an ablation laser (to generate endothelial injury), an image intensifier, and a camera [75]. Endothelial injury and thrombosis can also be induced by mechanical or chemical means, but laser injury offers the advantage of greater temporal and spatial control than the other methods [75]. There are a number of different microscopes that may be used, depending on the type of imaging required, which is in large part dictated by the depth of penetration required by type of tissue being examined, see Masedunskas et al. [69]. Recommendations for camera characteristics include that they be sensitive enough to detect the lowest expected fluorescent signals and to be able to reset fast enough to allow frame rates of 10 per second [75]. For further details on methods of performing intravital microscopy to study platelet function, see Stalker et al. [9], Herr et al. [11], and Falati et al. [70].

Intravital microscopy has several applications to identify underlying mechanisms of disease and new aspects of platelet biology. A few illustrative examples of the value

Intravital microscopy is a well-established research tool that has been increasingly applied to study platelets, real-time, *in vivo*. In this technique, the role of platelets in thrombus formation and other cellular activities such as interactions with leukocytes can be directly observed by the surgical creation of "imaging window" in anesthetized rat or mouse models. The major advantage of this approach is that it allows for observation of platelets in an environment that most closely matches what occurs *in vivo* in humans compared to other assays of platelet function. Imaging cells in living organisms has been practiced since the early twentieth century [67]. However, it was not until recently with the development of multiphoton or non-linear optical microscopy that has allowed for imaging in tissues at deeper depths (up to 1 mm) whereas conventional confocal microscopy can only be used up to depths of 50–60 μm [68, 69]. Lastly, with the advent of advanced computational power and digital image acquisition technology integrating with microscopes, quantitative analysis of fluorescent markers and other image properties can now be performed to more comprehensively evaluate properties of clot formation,

## **5. Intravital microscopy: imaging thrombus formation and platelet function** *in vivo*

## **5.1 Overview**

*Platelets*

the absence of dense granules on EM is criteria for diagnosis [17], while Chediak-Higashi syndrome can be confirmed by observing decreased dense bodies, normal alpha granules, and giant cytoplasmic inclusions [46]. While the clinical use of EM is not widely available, it remains an important tool for the diagnosis of rare, but important platelet disorders described above, as well as for several others [15, 46]. In addition to the current clinical applications of platelet EM for the diagnosis of platelet disorders, emerging research using cryo-ET shows promise for expanding our understanding of the role of platelets in a range of disease states including cancer [61], thrombo-inflammatory conditions, and for monitoring the quality of blood banking strategies for platelets [51, 58]. In a recent investigation using cryo-ET of platelets from patients with ovarian cancer, the authors identified important morphological changes in nine parameters between those with and without malignancy, including shortened microtubules, and increases in both size and number of platelet mitochondria [61]. These differences were then incorporated into a prediction model which accurately identified 20 out of 23 patients with ovarian cancer,

demonstrating exciting potential for future diagnostic purposes.

**4.5 Current limitations and future directions in electron microscopy**

Despite its clear potential as both a research and clinical tool, EM has important drawbacks. In addition to high costs, it requires specialized equipment and highly trained personnel, and therefore is neither widely available nor easy to automate. Furthermore, from a technical perspective, sample thickness is limited, as resolution dramatically decreases with increasing diameters, though this presents more of a challenge for imaging larger cells than it does for platelets [41]. Further, if an intracellular structure takes on an unexpected shape or localization, it can be very difficult to identify without the aid of immuno-labeling, which is not always feasible. Currently, techniques to label intracellular structures require fluorescent tagging and light microscopy, which can be correlated to electron microscopy images (correlative light and electron microscopy or CLEM). These have been improved upon with the use of super-resolution light microscopies as described above, but still does not reach the resolution of EM (super-resolution correlated light and electron microscopy) [35, 36, 41, 64]. However, emerging techniques using cloneable, genetic, electron dense markers, for direct localization with EM could have important applications if used with EM of platelets [65, 66]. Lastly, expanding the use of EM for diagnostic purposes, not just of rare platelet disorders, but in other conditions which affect platelets such as malignancy may provide important insights into disease processes as well as novel diagnostic

hemostatic plug's architecture [63].

Electron microscopy has also been used to characterize the properties of platelet microparticles, patterns of platelet activation, as well as the structure of platelet plug formation in *in vivo* models [18, 58, 62, 63]. For example, Ponomareva et al used both SEM and TEM to study the generation of microparticles from platelets in response to stimulation from adenosine diphosphate, arachidonic acid, and thrombin, and found that many microparticles were in fact smaller (less than 300 nm) than those typically able to be detected by flow cytometry [18]. Furthermore, they found that microparticles isolated from patients with conditions such as disseminated intravascular coagulation, heparin induced thrombocytopenia, or lupus were larger and displayed pathologic properties [18]. Lastly, in an *in vivo* mouse model of vascular injury, Tomaiuolo et al used a combination of fluorescence microscopy and SEM to detail different levels of platelet activation within a forming thrombus, and the effects of antiplatelet agents on the

**10**

approaches [61].

Intravital microscopy is a well-established research tool that has been increasingly applied to study platelets, real-time, *in vivo*. In this technique, the role of platelets in thrombus formation and other cellular activities such as interactions with leukocytes can be directly observed by the surgical creation of "imaging window" in anesthetized rat or mouse models. The major advantage of this approach is that it allows for observation of platelets in an environment that most closely matches what occurs *in vivo* in humans compared to other assays of platelet function. Imaging cells in living organisms has been practiced since the early twentieth century [67]. However, it was not until recently with the development of multiphoton or non-linear optical microscopy that has allowed for imaging in tissues at deeper depths (up to 1 mm) whereas conventional confocal microscopy can only be used up to depths of 50–60 μm [68, 69]. Lastly, with the advent of advanced computational power and digital image acquisition technology integrating with microscopes, quantitative analysis of fluorescent markers and other image properties can now be performed to more comprehensively evaluate properties of clot formation, and distinctive platelet activation and signaling patterns [9, 11, 70, 71].

#### **5.2 Choice of site and imaging technique**

The cremaster muscle and mesenteric veins of mice are the most commonly used imaging windows for intravital microscopy, but other possible sites include carotid artery, brain, skin, bones, or liver [11, 72, 73]. In order for visualization and subsequent quantitative analyses, platelets must be labeled with fluorescent antibodies or genetically modified mouse models which express fluorescent platelet proteins. Both of these approaches provide adequate capacity to visualize platelets *in vivo*, however, at least for some of the genetic approaches in mice, there may be untoward effects on platelet aggregation compared to antibody labeling approaches [74]. While any vascular bed theoretically may be used to study platelet function, the primary limitations include the anatomic challenges of isolating the vessels while minimizing trauma and inflammation, as well as vessel caliber, since it must be sufficiently transparent for light penetration [75]. Pros and cons of each anatomic site are summarized below (**Table 1**).

The essential imaging equipment includes a microscope, an illumination light source for fluorescence excitation, an ablation laser (to generate endothelial injury), an image intensifier, and a camera [75]. Endothelial injury and thrombosis can also be induced by mechanical or chemical means, but laser injury offers the advantage of greater temporal and spatial control than the other methods [75]. There are a number of different microscopes that may be used, depending on the type of imaging required, which is in large part dictated by the depth of penetration required by type of tissue being examined, see Masedunskas et al. [69]. Recommendations for camera characteristics include that they be sensitive enough to detect the lowest expected fluorescent signals and to be able to reset fast enough to allow frame rates of 10 per second [75]. For further details on methods of performing intravital microscopy to study platelet function, see Stalker et al. [9], Herr et al. [11], and Falati et al. [70].

#### **5.3 Applications and examples**

Intravital microscopy has several applications to identify underlying mechanisms of disease and new aspects of platelet biology. A few illustrative examples of the value


*\*See Merrill-Skoloff et al. [75], Falati et al. [70], and Masedunskas et al. [69] for further descriptions of approaches to each site.*

#### **Table 1.**

*Anatomic locations for intravital microscopy, advantages and limitations.\**

and versatility of the technique include studies demonstrating mechanisms of microvascular thrombosis, platelet production, and for examining platelet leukocyte interactions [19, 72, 73, 76]. For example, in a murine model of arteriolar thrombosis, Lu et al employed cranial intravital microscopy to study anti-platelet properties of caffeic acid (**Figure 6**) [72]. It can also be used to better characterize platelet interactions with other cell types. Using liver intravital microscopy to examine pathways involved in steatohepatitis, Malehmir et al. showed that platelet colonization depended on Kupffer cells and CD44 binding, and that treatment with anti-platelet therapy reduced infiltration of platelets into liver cells as well as platelet immune interactions and trafficking, ultimately leading to attenuation of liver damage [73]. Lastly, the identification of the lung microvasculature as a site of platelet production is another remarkable discovery made possible by the use of intravital microscopy [19].

#### **5.4 Limitations and future advances**

The main limitations of intravital microscopy include the depth of penetration depending on the type of microscope used, technical challenges, heterogeneous features and low transparency of certain tissues, and limitations on the number of fluorescent tags that can be used simultaneously [68, 69, 75]. Future advances may include incorporation of correlative light and electron microscopy (CLEM) to intravital approaches, technical advances in miniaturization of lenses and imaging equipment, and improvements in the array of genetic and molecular markers used to track platelet-cellular and platelet-ultrastructural processes [68].

**13**

and function.

**Conflict of interest**

Dr. Kornblith and Dr. Matthay have nothing to disclose.

*Platelet Imaging*

**Figure 6.**

*licenses/by-sa/4.0/deed.en).*

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

**6. Conclusions and future directions**

An increasingly versatile armamentarium of imaging modalities is available for research and diagnostic purposes to study platelets [11, 61, 68, 71]. We anticipate these will continue to play instrumental roles in further discoveries in the field of platelet biology, revealing new insights into the dynamics of platelet signaling, ultrastructure, and function. Understanding how to utilize these evolving imaging technologies to best suit the visualization of platelets is essential. Given the inherently highly reactive nature of platelets, newer approaches that cause minimal manipulation and processing offer significant advantages, including intravital microscopy for studying the roles of platelets roles in thrombus formation and cellular interactions [70], and cryogenic electron tomography which preserves platelets in a near native state for ultrastructural analysis [61]. Important advances continue to be made in labeling approaches that can be paired with imaging modalities, including correlative light and electron microscopy, and genetically inducible fluorescent or electron dense markers [34, 65, 66, 77]. In addition to the technical advances in our imaging capabilities, their integration with advanced statistical and computational power will continue to reveal new opportunities to analyze and better understand platelet structure

*Cranial intravital microscopy of thrombus formation in a mouse model. (A) Representative images of thrombus formation in mouse cerebral arterioles induced by photochemical injury in different conditions and at different time points. In order to compare the activities of CA and clopidogrel at a similar blood concentration, 5 mg/kg of CA (MW: 180.16) and 12 mg/kg of clopidogrel (MW: 419.90) were continuously infused through the catheterized femoral vein starting from 20 min prior to the vessel wall injury. This resulted in approximately the same blood concentration of CA and clopidogrel in mice* in vivo*. Arrows indicate irreversible arteriolar vessel occlusion. (B) Dot plots of arteriolar occlusion time from five to six experiments.* p *< 0.05* vs *Model group.* Abbreviations*: CA, caffeic acid; MW, molecular weight. Figure and caption reproduced from Lu et al. [76], under the Creative Commons Attribution 4.0 International License (https://creativecommons.org/*

#### **Figure 6.**

*Platelets*

Cremaster Muscle

Carotid Artery

*to each site.*

**Table 1.**

Mesentery Mesentery

Ear Ear is shaved and

artery

Brain Cranial window on

Liver Lateral abdominal

**12**

and versatility of the technique include studies demonstrating mechanisms of microvascular thrombosis, platelet production, and for examining platelet leukocyte interactions [19, 72, 73, 76]. For example, in a murine model of arteriolar thrombosis, Lu et al employed cranial intravital microscopy to study anti-platelet properties of caffeic acid (**Figure 6**) [72]. It can also be used to better characterize platelet interactions with other cell types. Using liver intravital microscopy to examine pathways involved in steatohepatitis, Malehmir et al. showed that platelet colonization depended on Kupffer cells and CD44 binding, and that treatment with anti-platelet therapy reduced infiltration of platelets into liver cells as well as platelet immune interactions and trafficking, ultimately leading to attenuation of liver damage [73]. Lastly, the identification of the lung microvasculature as a site of platelet production is another

*\*See Merrill-Skoloff et al. [75], Falati et al. [70], and Masedunskas et al. [69] for further descriptions of approaches* 

**Site General approach Advantages Limitations**

injury

Quick preparation (5–7 min) Thin, transparent Multiple vessels can be studied simultaneously

Mesenteric vessels large enough for easy ligation or glass pipette mechanical

Vessel large enough for Doppler flow monitoring

Amenable to conventional confocal microscopy

Good for studying microvasculature

Vessels are too small to assess

Older mice (>3–4 weeks) have too much fat around vessels to allow for brightfield microscopy Fewer vessels can be studied

reliable injury models due to variation in vascular

Only amenable to fluorescent microscopy, no bright field images can be obtained

Technically challenging

Technically challenging

flow by Doppler

simultaneously Greater trauma during sample preparation

anatomy

Simple preparation Difficult to produce

Muscle is exteriorized through scrotal incision in mice

exteriorized through midline incision

trans-illuminated to identify vessels

Neck incision with dissection of carotid

dorsal surface of skull

*Anatomic locations for intravital microscopy, advantages and limitations.\**

incision with liver exteriorization

remarkable discovery made possible by the use of intravital microscopy [19].

to track platelet-cellular and platelet-ultrastructural processes [68].

The main limitations of intravital microscopy include the depth of penetration depending on the type of microscope used, technical challenges, heterogeneous features and low transparency of certain tissues, and limitations on the number of fluorescent tags that can be used simultaneously [68, 69, 75]. Future advances may include incorporation of correlative light and electron microscopy (CLEM) to intravital approaches, technical advances in miniaturization of lenses and imaging equipment, and improvements in the array of genetic and molecular markers used

**5.4 Limitations and future advances**

*Cranial intravital microscopy of thrombus formation in a mouse model. (A) Representative images of thrombus formation in mouse cerebral arterioles induced by photochemical injury in different conditions and at different time points. In order to compare the activities of CA and clopidogrel at a similar blood concentration, 5 mg/kg of CA (MW: 180.16) and 12 mg/kg of clopidogrel (MW: 419.90) were continuously infused through the catheterized femoral vein starting from 20 min prior to the vessel wall injury. This resulted in approximately the same blood concentration of CA and clopidogrel in mice* in vivo*. Arrows indicate irreversible arteriolar vessel occlusion. (B) Dot plots of arteriolar occlusion time from five to six experiments.* p *< 0.05* vs *Model group.* Abbreviations*: CA, caffeic acid; MW, molecular weight. Figure and caption reproduced from Lu et al. [76], under the Creative Commons Attribution 4.0 International License (https://creativecommons.org/ licenses/by-sa/4.0/deed.en).*

## **6. Conclusions and future directions**

An increasingly versatile armamentarium of imaging modalities is available for research and diagnostic purposes to study platelets [11, 61, 68, 71]. We anticipate these will continue to play instrumental roles in further discoveries in the field of platelet biology, revealing new insights into the dynamics of platelet signaling, ultrastructure, and function. Understanding how to utilize these evolving imaging technologies to best suit the visualization of platelets is essential. Given the inherently highly reactive nature of platelets, newer approaches that cause minimal manipulation and processing offer significant advantages, including intravital microscopy for studying the roles of platelets roles in thrombus formation and cellular interactions [70], and cryogenic electron tomography which preserves platelets in a near native state for ultrastructural analysis [61]. Important advances continue to be made in labeling approaches that can be paired with imaging modalities, including correlative light and electron microscopy, and genetically inducible fluorescent or electron dense markers [34, 65, 66, 77]. In addition to the technical advances in our imaging capabilities, their integration with advanced statistical and computational power will continue to reveal new opportunities to analyze and better understand platelet structure and function.

## **Conflict of interest**

Dr. Kornblith and Dr. Matthay have nothing to disclose.

## **Funding**

Dr. Kornblith is supported by NIH 1K23GM130892-01; Dr. Matthay is supported by The National Center for Advancing Translational Sciences of the NIH award 5TL1TR001871-04.

## **Other declarations**

Human and Animal Rights Informed Consent: This study does not contain any studies with human or animal subjects performed by any of the authors.

## **Author details**

Zachary A. Matthay\* and Lucy Zumwinkle Kornblith\* Division of General Surgery, Trauma and Surgical Critical Care, Zuckerberg San Francisco General Hospital, San Francisco, CA, USA

\*Address all correspondence to: zachary.matthay@ucsf.edu and lucy.kornblith@ucsf.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**15**

*Platelet Imaging*

**References**

2015;**26**(4):285

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

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response and its relationship to the platelet-signaling network. Blood.

[10] Cramer EM, Meyer D, le Menn R, Breton-Gorius J. Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies. Blood. 1985;**66**(3):710-713

[11] Herr N, Mauler M, Bode C, Duerschmied D. Intravital microscopy of leukocyte-endothelial and plateletleukocyte interactions in mesenterial veins in mice. Journal of Visualized Experiments. 2015;**102**:e53077

[12] Kerrigan SW, Clarke N, Loughman A, Meade G, Foster TJ,

Cox D. Molecular basis for *Staphylococcus aureus*-mediated platelet aggregate formation under arterial shear in vitro. Arteriosclerosis, Thrombosis, and Vascular Biology.

[13] White JG. Morphological studies of platelets and platelet reactions. Vox Sanguinis. 1981;**40**(Suppl 1):8-17

[14] White JG. Electron microscopy methods for studying platelet structure and function. Methods in Molecular

[15] Clauser S, Cramer-Borde E. Role of platelet electron microscopy in the diagnosis of platelet disorders. Seminars

in Thrombosis and Hemostasis.

[16] White JG. Use of the electron microscope for diagnosis of platelet disorders. Seminars in Thrombosis and Hemostasis. 1998;**24**(2):163-168

[17] Witkop CJ, Krumwiede M,

Sedano H, White JG. Reliability of absent platelet dense bodies as a diagnostic criterion for Hermansky-Pudlak

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Biology. 2004;**272**:47-63

2009;**35**(2):213-223

2013;**121**(10):1875-1885

[2] Nachman RL, Rafii S. Platelets, petechiae, and preservation of the vascular wall. The New England Journal of Medicine. 2008;**359**(12):1261-1270

[3] Kerrigan SW, Poole A. Focusing on the role of platelets in immune defence against invading pathogens. Platelets.

[4] Hoffman M, Monroe DM 3rd. A cellbased model of hemostasis. Thrombosis and Haemostasis. 2001;**85**(6):958-965

Zimmerman GA. Platelets as cellular effectors of inflammation in vascular diseases. Circulation Research.

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Experimental Pharmacology. 2012;**210**:

[7] van Nispen tot Pannerden H, de Haas F, Geerts W, Posthuma G, van Dijk S, Heijnen HF. The platelet interior revisited: Electron tomography reveals tubular alpha-granule subtypes. Blood.

[8] Barendrecht AD, Verhoef JJF,

[9] Stalker TJ, Traxler EA, Wu J, Wannemacher KM, Cermignano SL, Voronov R, et al. Hierarchical organization in the hemostatic

Pignatelli S, Pasterkamp G, Heijnen HFG, Maas C. Live-cell imaging of platelet degranulation and secretion under flow. Journal of Visualized Experiments.

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3-22

## **References**

*Platelets*

**Funding**

5TL1TR001871-04.

**Other declarations**

**14**

**Author details**

and lucy.kornblith@ucsf.edu

provided the original work is properly cited.

Zachary A. Matthay\* and Lucy Zumwinkle Kornblith\*

Francisco General Hospital, San Francisco, CA, USA

\*Address all correspondence to: zachary.matthay@ucsf.edu

Division of General Surgery, Trauma and Surgical Critical Care, Zuckerberg San

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Dr. Kornblith is supported by NIH 1K23GM130892-01; Dr. Matthay is supported

Human and Animal Rights Informed Consent: This study does not contain any

studies with human or animal subjects performed by any of the authors.

by The National Center for Advancing Translational Sciences of the NIH award

[1] Vieira-de-Abreu A, Campbell RA, Weyrich AS, Zimmerman GA. Platelets: Versatile effector cells in hemostasis, inflammation, and the immune continuum. Seminars in Immunopathology. 2012;**34**(1):5-30

[2] Nachman RL, Rafii S. Platelets, petechiae, and preservation of the vascular wall. The New England Journal of Medicine. 2008;**359**(12):1261-1270

[3] Kerrigan SW, Poole A. Focusing on the role of platelets in immune defence against invading pathogens. Platelets. 2015;**26**(4):285

[4] Hoffman M, Monroe DM 3rd. A cellbased model of hemostasis. Thrombosis and Haemostasis. 2001;**85**(6):958-965

[5] Rondina MT, Weyrich AS, Zimmerman GA. Platelets as cellular effectors of inflammation in vascular diseases. Circulation Research. 2013;**112**(11):1506-1519

[6] Thon JN, Italiano JE. Platelets: Production, morphology and ultrastructure. Handbook of Experimental Pharmacology. 2012;**210**: 3-22

[7] van Nispen tot Pannerden H, de Haas F, Geerts W, Posthuma G, van Dijk S, Heijnen HF. The platelet interior revisited: Electron tomography reveals tubular alpha-granule subtypes. Blood. 2010;**116**(7):1147-1156

[8] Barendrecht AD, Verhoef JJF, Pignatelli S, Pasterkamp G, Heijnen HFG, Maas C. Live-cell imaging of platelet degranulation and secretion under flow. Journal of Visualized Experiments. 2017;**125**:e-55658

[9] Stalker TJ, Traxler EA, Wu J, Wannemacher KM, Cermignano SL, Voronov R, et al. Hierarchical organization in the hemostatic

response and its relationship to the platelet-signaling network. Blood. 2013;**121**(10):1875-1885

[10] Cramer EM, Meyer D, le Menn R, Breton-Gorius J. Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies. Blood. 1985;**66**(3):710-713

[11] Herr N, Mauler M, Bode C, Duerschmied D. Intravital microscopy of leukocyte-endothelial and plateletleukocyte interactions in mesenterial veins in mice. Journal of Visualized Experiments. 2015;**102**:e53077

[12] Kerrigan SW, Clarke N, Loughman A, Meade G, Foster TJ, Cox D. Molecular basis for *Staphylococcus aureus*-mediated platelet aggregate formation under arterial shear in vitro. Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;**28**(2):335-340

[13] White JG. Morphological studies of platelets and platelet reactions. Vox Sanguinis. 1981;**40**(Suppl 1):8-17

[14] White JG. Electron microscopy methods for studying platelet structure and function. Methods in Molecular Biology. 2004;**272**:47-63

[15] Clauser S, Cramer-Borde E. Role of platelet electron microscopy in the diagnosis of platelet disorders. Seminars in Thrombosis and Hemostasis. 2009;**35**(2):213-223

[16] White JG. Use of the electron microscope for diagnosis of platelet disorders. Seminars in Thrombosis and Hemostasis. 1998;**24**(2):163-168

[17] Witkop CJ, Krumwiede M, Sedano H, White JG. Reliability of absent platelet dense bodies as a diagnostic criterion for Hermansky-Pudlak

syndrome. American Journal of Hematology. 1987;**26**(4):305-311

[18] Ponomareva AA, Nevzorova TA, Mordakhanova ER, Andrianova IA, Rauova L, Litvinov RI, et al. Intracellular origin and ultrastructure of plateletderived microparticles. Journal of Thrombosis and Haemostasis. 2017;**15**(8):1655-1667

[19] Lefrancais E, Ortiz-Munoz G, Caudrillier A, Mallavia B, Liu F, Sayah DM, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature. 2017;**544**(7648):105-109

[20] Ribatti D, Crivellato E. Giulio Bizzozero and the discovery of platelets. Leukemia Research. 2007;**31**(10):1339-1341

[21] Greinacher A, Pecci A, Kunishima S, Althaus K, Nurden P, Balduini CL, et al. Diagnosis of inherited platelet disorders on a blood smear: A tool to facilitate worldwide diagnosis of platelet disorders. Journal of Thrombosis and Haemostasis. 2017;**15**(7):1511-1521

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[23] Sandrock K, Knofler R, Greinacher A, Furll B, Gerisch S, Schuler U, et al. Novel mutation in Bernard-Soulier syndrome. Transfusion Medicine and Hemotherapy. 2010;**37**(5):278-284

[24] Aslan JE, Itakura A, Gertz JM, McCarty OJ. Platelet shape change and spreading. Methods in Molecular Biology. 2012;**788**:91-100

[25] Thomas SG, Calaminus SD, Auger JM, Watson SP, Machesky LM. Studies on the actin-binding protein HS1 in platelets. BMC Cell Biology. 2007;**8**:46

[26] Eshel-Green T, Berny MA, Conley RB, McCarty OJ. Effect of sex difference on platelet adhesion, spreading and aggregate formation under flow. Thrombosis and Haemostasis. 2009;**102**(5):958-965

[27] Calaminus SD, Thomas S, McCarty OJ, Machesky LM, Watson SP. Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading. Journal of Thrombosis and Haemostasis. 2008;**6**(11):1944-1952

[28] Calaminus SD, McCarty OJ, Auger JM, Pearce AC, Insall RH, Watson SP, et al. A major role for scar/ WAVE-1 downstream of GPVI in platelets. Journal of Thrombosis and Haemostasis. 2007;**5**(3):535-541

[29] Tunstromer K, Faxalv L, Boknas N, Lindahl TL. Quantification of platelet contractile movements during thrombus formation. Thrombosis and Haemostasis. 2018;**118**(9):1600-1611

[30] Sakariassen KS, Aarts PA, de Groot PG, Houdijk WP, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. The Journal of Laboratory and Clinical Medicine. 1983;**102**(4):522-535

[31] Claesson K, Lindahl TL, Faxalv L. Counting the platelets: A robust and sensitive quantification method for thrombus formation. Thrombosis and Haemostasis. 2016;**115**(6):1178-1190

[32] Roest M, Reininger A, Zwaginga JJ, King MR, Heemskerk JW. Biorheology subcommittee of the SSCotI. Flow chamber-based assays to measure thrombus formation in vitro: Requirements for standardization. Journal of Thrombosis and Haemostasis. 2011;**9**(11):2322-2324

**17**

*Platelet Imaging*

229-242

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

structural biology in situ. Annual Review of Biochemistry. 2017;**86**:

[42] White JG. The dense bodies of human platelets. Origin of serotonin storage particles from platelet granules. The American Journal of Pathology.

[43] White JG. Ultrastructural defects in congenital disorders of platelet function. Annals of the New York Academy of Sciences. 1972;**201**:205-233

[44] White JG. Identification of platelet secretion in the electron microscope. Series Haematologica. 1973;**6**(3):429-459

Ultrastructural features of abnormal blood platelets. A review. The American

[46] White JG. Platelet microtubules and giant granules in the Chediak-Higashi syndrome. The American Journal of Medical Technology. 1978;**44**(4):273-278

[47] White JG. Ultrastructural studies of the gray platelet syndrome. The American Journal of Pathology.

[48] White JG. An overview of platelet structural physiology. Scanning Microscopy. 1987;**1**(4):1677-1700

Microtubules and fibrils in spread-out platelets. Nouvelle Revue Française d'Hématologie. 1965;**5**(4):657-662

[50] Bessis M, Dreyfus B, Breton-Gorius J, Sultan C. Electron microscopic study of 11 cases of refractory anemias with multiple enzymopathies. Nouvelle Revue Française d'Hématologie. 1969;**9**(1):

[51] Neumüller J, Ellinger A, Wagner T. Transmission Electron Microscopy of

[49] Bessis M, Breton-Gorius J.

1979;**95**(2):445-462

Journal of Pathology. 1976;**83**(3):

[45] White JG, Gerrard JM.

589-632

87-104

873-896

1968;**53**(5):791-808

[33] Van Kruchten R, Cosemans JM, Heemskerk JW. Measurement of whole blood thrombus formation using parallel-plate flow chambers—A practical guide. Platelets. 2012;**23**(3):

[34] Knight AE, Gomez K, Cutler DF. Super-resolution microscopy in the diagnosis of platelet granule disorders.

Expert Review of Hematology.

[35] Westmoreland D, Shaw M, Grimes W, Metcalf DJ, Burden JJ, Gomez K, et al. Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders. Journal of Thrombosis and Haemostasis.

[36] Meixner AJ. The Nobel prize in chemistry 2014 for the development of super-resolved fluorescence microscopy. Analytical and Bioanalytical Chemistry.

[37] Schermelleh L, Ferrand A, Huser T, Eggeling C, Sauer M, Biehlmaier O, et al. Super-resolution microscopy demystified. Nature Cell Biology.

[38] Schermelleh L, Heintzmann R, Leonhardt H. A guide to super-resolution fluorescence microscopy. The Journal of Cell Biology. 2010;**190**(2):165-175

[39] Poulter NS, Pollitt AY, Davies A, Malinova D, Nash GB, Hannon MJ, et al. Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex. Nature Communications.

[40] Ruska E. Nobel lecture. The

Reports. 1987;**7**(8):607-629

development of the electron microscope and of electron microscopy. Bioscience

[41] Oikonomou CM, Jensen GJ. Cellular electron cryotomography: Toward

2017;**10**(5):375-381

2016;**14**(4):839-849

2015;**407**(7):1797-1800

2019;**21**(1):72-84

2015;**6**:7254

#### *Platelet Imaging DOI: http://dx.doi.org/10.5772/intechopen.91736*

*Platelets*

syndrome. American Journal of Hematology. 1987;**26**(4):305-311

2017;**15**(8):1655-1667

2017;**544**(7648):105-109

[18] Ponomareva AA, Nevzorova TA, Mordakhanova ER, Andrianova IA, Rauova L, Litvinov RI, et al. Intracellular origin and ultrastructure of plateletderived microparticles. Journal of Thrombosis and Haemostasis.

[26] Eshel-Green T, Berny MA, Conley RB, McCarty OJ. Effect of sex difference on platelet adhesion, spreading and aggregate formation under flow. Thrombosis and Haemostasis. 2009;**102**(5):958-965

[27] Calaminus SD, Thomas S,

[28] Calaminus SD, McCarty OJ, Auger JM, Pearce AC, Insall RH, Watson SP, et al. A major role for scar/ WAVE-1 downstream of GPVI in platelets. Journal of Thrombosis and Haemostasis. 2007;**5**(3):535-541

[29] Tunstromer K, Faxalv L, Boknas N, Lindahl TL. Quantification of platelet

thrombus formation. Thrombosis and Haemostasis. 2018;**118**(9):1600-1611

[31] Claesson K, Lindahl TL, Faxalv L. Counting the platelets: A robust and sensitive quantification method for thrombus formation. Thrombosis and Haemostasis. 2016;**115**(6):1178-1190

[32] Roest M, Reininger A, Zwaginga JJ, King MR, Heemskerk JW. Biorheology subcommittee of the SSCotI. Flow chamber-based assays to measure thrombus formation in vitro: Requirements for standardization. Journal of Thrombosis and Haemostasis.

contractile movements during

[30] Sakariassen KS, Aarts PA, de Groot PG, Houdijk WP, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. The Journal of Laboratory and Clinical Medicine.

1983;**102**(4):522-535

2011;**9**(11):2322-2324

2008;**6**(11):1944-1952

McCarty OJ, Machesky LM, Watson SP. Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading. Journal of Thrombosis and Haemostasis.

[19] Lefrancais E, Ortiz-Munoz G, Caudrillier A, Mallavia B, Liu F, Sayah DM, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature.

[20] Ribatti D, Crivellato E. Giulio Bizzozero and the discovery of platelets. Leukemia Research. 2007;**31**(10):1339-1341

[22] Balduini CL, Pecci A, Noris P. Diagnosis and management of inherited

[23] Sandrock K, Knofler R, Greinacher A, Furll B, Gerisch S, Schuler U, et al. Novel mutation in Bernard-Soulier syndrome. Transfusion Medicine and Hemotherapy. 2010;**37**(5):278-284

[24] Aslan JE, Itakura A, Gertz JM, McCarty OJ. Platelet shape change and spreading. Methods in Molecular

[25] Thomas SG, Calaminus SD, Auger JM, Watson SP, Machesky LM. Studies on the actin-binding protein HS1 in platelets. BMC Cell Biology.

Biology. 2012;**788**:91-100

thrombocytopenias. Seminars in Thrombosis and Hemostasis.

2013;**39**(2):161-171

[21] Greinacher A, Pecci A, Kunishima S, Althaus K, Nurden P, Balduini CL, et al. Diagnosis of inherited platelet disorders on a blood smear: A tool to facilitate worldwide diagnosis of platelet disorders. Journal of Thrombosis and Haemostasis. 2017;**15**(7):1511-1521

**16**

2007;**8**:46

[33] Van Kruchten R, Cosemans JM, Heemskerk JW. Measurement of whole blood thrombus formation using parallel-plate flow chambers—A practical guide. Platelets. 2012;**23**(3): 229-242

[34] Knight AE, Gomez K, Cutler DF. Super-resolution microscopy in the diagnosis of platelet granule disorders. Expert Review of Hematology. 2017;**10**(5):375-381

[35] Westmoreland D, Shaw M, Grimes W, Metcalf DJ, Burden JJ, Gomez K, et al. Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders. Journal of Thrombosis and Haemostasis. 2016;**14**(4):839-849

[36] Meixner AJ. The Nobel prize in chemistry 2014 for the development of super-resolved fluorescence microscopy. Analytical and Bioanalytical Chemistry. 2015;**407**(7):1797-1800

[37] Schermelleh L, Ferrand A, Huser T, Eggeling C, Sauer M, Biehlmaier O, et al. Super-resolution microscopy demystified. Nature Cell Biology. 2019;**21**(1):72-84

[38] Schermelleh L, Heintzmann R, Leonhardt H. A guide to super-resolution fluorescence microscopy. The Journal of Cell Biology. 2010;**190**(2):165-175

[39] Poulter NS, Pollitt AY, Davies A, Malinova D, Nash GB, Hannon MJ, et al. Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex. Nature Communications. 2015;**6**:7254

[40] Ruska E. Nobel lecture. The development of the electron microscope and of electron microscopy. Bioscience Reports. 1987;**7**(8):607-629

[41] Oikonomou CM, Jensen GJ. Cellular electron cryotomography: Toward

structural biology in situ. Annual Review of Biochemistry. 2017;**86**: 873-896

[42] White JG. The dense bodies of human platelets. Origin of serotonin storage particles from platelet granules. The American Journal of Pathology. 1968;**53**(5):791-808

[43] White JG. Ultrastructural defects in congenital disorders of platelet function. Annals of the New York Academy of Sciences. 1972;**201**:205-233

[44] White JG. Identification of platelet secretion in the electron microscope. Series Haematologica. 1973;**6**(3):429-459

[45] White JG, Gerrard JM. Ultrastructural features of abnormal blood platelets. A review. The American Journal of Pathology. 1976;**83**(3): 589-632

[46] White JG. Platelet microtubules and giant granules in the Chediak-Higashi syndrome. The American Journal of Medical Technology. 1978;**44**(4):273-278

[47] White JG. Ultrastructural studies of the gray platelet syndrome. The American Journal of Pathology. 1979;**95**(2):445-462

[48] White JG. An overview of platelet structural physiology. Scanning Microscopy. 1987;**1**(4):1677-1700

[49] Bessis M, Breton-Gorius J. Microtubules and fibrils in spread-out platelets. Nouvelle Revue Française d'Hématologie. 1965;**5**(4):657-662

[50] Bessis M, Dreyfus B, Breton-Gorius J, Sultan C. Electron microscopic study of 11 cases of refractory anemias with multiple enzymopathies. Nouvelle Revue Française d'Hématologie. 1969;**9**(1): 87-104

[51] Neumüller J, Ellinger A, Wagner T. Transmission Electron Microscopy of

Platelets from Apheresis and Buffy-Coat-Derived Platelet Concentrates, the Transmission Electron Microscope— Theory and Applications. Rijeka: IntechOpen; 2015. Available from: https://www.intechopen.com/ books/the-transmission-electronmicroscope-theory-and-applications/ transmission-electron-microscopy-ofplatelets-from-apheresis-and-buffycoat-derived-platelet-concent

[52] Bentfeld-Barker ME, Bainton DF. Identification of primary lysosomes in human megakaryocytes and platelets. Blood. 1982;**59**(3):472-481

[53] Boudreau LH, Duchez AC, Cloutier N, Soulet D, Martin N, Bollinger J, et al. Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation. Blood. 2014;**124**(14):2173-2183

[54] Marcoux G, Magron A, Sut C, Laroche A, Laradi S, Hamzeh-Cognasse H, et al. Plateletderived extracellular vesicles convey mitochondrial DAMPs in platelet concentrates and their levels are associated with adverse reactions. Transfusion. 2019;**59**(7):2403-2414

[55] Heijnen HF, Oorschot V, Sixma JJ, Slot JW, James DE. Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from alphagranules to the cell surface. The Journal of Cell Biology. 1997;**138**(2):323-330

[56] Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, et al. Cryo-electron microscopy of vitrified specimens. Quarterly Reviews of Biophysics. 1988;**21**(2):129-228

[57] Koster AJ, Grimm R, Typke D, Hegerl R, Stoschek A, Walz J, et al. Perspectives of molecular and cellular electron tomography. Journal of Structural Biology. 1997;**120**(3):276-308 [58] Nemeüller J, Meisslitzer-Ruppitsch C, Ellinger A, Pavelka M, Jungbauer C, Renz R, et al. Monitoring of platelet activation in platelet concentrates using transmission electron microscopy. Transfusion Medicine and Hemotherapy. 2013;**40**(2):101-107

[59] Raccuglia G. Gray platelet syndrome. A variety of qualitative platelet disorder. The American Journal of Medicine. 1971;**51**(6):818-828

[60] Drouin A, Favier R, Masse JM, Debili N, Schmitt A, Elbim C, et al. Newly recognized cellular abnormalities in the gray platelet syndrome. Blood. 2001;**98**(5):1382-1391

[61] Wang R, Stone RL, Kaelber JT, Rochat RH, Nick AM, Vijayan KV, et al. Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**(46):14266-14271

[62] Ramtoola Z, Lyons P, Keohane K, Kerrigan SW, Kirby BP, Kelly JG. Investigation of the interaction of biodegradable micro- and nanoparticulate drug delivery systems with platelets. The Journal of Pharmacy and Pharmacology. 2011;**63**(1):26-32

[63] Tomaiuolo M, Matzko CN, Poventud-Fuentes I, Weisel JW, Brass LF, Stalker TJ. Interrelationships between structure and function during the hemostatic response to injury. Proceedings of the National Academy of Sciences of the United States of America. 2019;**116**(6):2243-2252

[64] Chang YW, Chen S, Tocheva EI, Treuner-Lange A, Lobach S, Sogaard-Andersen L, et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nature Methods. 2014;**11**(7):737-739

**19**

*Platelet Imaging*

2011;**19**(2):147-154

1931;**7**(1):31-61

2010;**133**(5):481-491

2012;**2**(5):143-157

[69] Masedunskas A, Milberg O,

Porat-Shliom N, Sramkova M, Wigand T, Amornphimoltham P, et al. Intravital microscopy: A practical guide on imaging intracellular structures in live animals. BioArchitecture.

[70] Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nature Medicine. 2002;**8**(10):1175-1181

Jasuja R, Furie B, Furie BC. Imaging fibrin formation and platelet and endothelial cell activation in vivo. Thrombosis and Haemostasis. 2011;**105**(5):776-782

[72] Adili R, Holinstat M. Formation and resolution of Pial microvascular thrombosis in a mouse model of

thrombotic thrombocytopenic Purpura. Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;**39**(9):1817-1830

[73] Malehmir M, Pfister D, Gallage S, Szydlowska M, Inverso D, Kotsiliti E,

[71] Bellido-Martin L, Chen V,

*DOI: http://dx.doi.org/10.5772/intechopen.91736*

et al. Platelet GPIbalpha is a mediator and potential interventional target for NASH and subsequent liver cancer. Nature Medicine. 2019;**25**(4):641-655

[74] Da Q, Derry PJ, Lam FW, Rumbaut RE. Fluorescent labeling of endogenous platelets for intravital microscopy: Effects on platelet function. Microcirculation. 2018;**25**(6):e12457

[75] Merrill-Skoloff G, Atkinson BT, Furie BC, Furie B. Real time in vivo imaging of platelets during thrombus formation. In: Michelson A, editor. Platelets. 3rd ed. Netherlands: Elsevier;

[76] Lu Y, Li Q, Liu YY, Sun K, Fan JY, Wang CS, et al. Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases. Scientific Reports. 2015;**5**:13824

[77] Chen D, Uhl CB, Bryant SC, Krumwiede M, Barness RL,

Olson MC, et al. Diagnostic laboratory standardization and validation of platelet transmission electron microscopy. Platelets. 2018;**29**(6):574-582

2013. pp. 635-652

[65] Wang Q, Mercogliano CP, Lowe J. A ferritin-based label for cellular electron cryotomography. Structure.

[66] Mercogliano CP, DeRosier DJ. Concatenated metallothionein as a clonable gold label for electron microscopy. Journal of Structural Biology. 2007;**160**(1):70-82

[67] Beck JS, Berg BN. The circulatory pattern in the islands of Langerhans. The American Journal of Pathology.

[68] Weigert R, Sramkova M, Parente L, Amornphimoltham P, Masedunskas A. Intravital microscopy: A novel tool to study cell biology in living animals. Histochemistry and Cell Biology.

#### *Platelet Imaging DOI: http://dx.doi.org/10.5772/intechopen.91736*

*Platelets*

Platelets from Apheresis and Buffy-Coat-Derived Platelet Concentrates, the Transmission Electron Microscope— Theory and Applications. Rijeka: IntechOpen; 2015. Available from: https://www.intechopen.com/ books/the-transmission-electronmicroscope-theory-and-applications/ transmission-electron-microscopy-ofplatelets-from-apheresis-and-buffycoat-derived-platelet-concent

[58] Nemeüller J, Meisslitzer-Ruppitsch C, Ellinger A, Pavelka M, Jungbauer C, Renz R, et al. Monitoring of platelet activation in platelet concentrates using transmission electron microscopy. Transfusion Medicine and Hemotherapy.

2013;**40**(2):101-107

2001;**98**(5):1382-1391

[62] Ramtoola Z, Lyons P,

of biodegradable micro- and

[63] Tomaiuolo M, Matzko CN,

2019;**116**(6):2243-2252

2014;**11**(7):737-739

Keohane K, Kerrigan SW, Kirby BP, Kelly JG. Investigation of the interaction

nanoparticulate drug delivery systems with platelets. The Journal of Pharmacy and Pharmacology. 2011;**63**(1):26-32

Poventud-Fuentes I, Weisel JW, Brass LF, Stalker TJ. Interrelationships between structure and function during the hemostatic response to injury. Proceedings of the National Academy of Sciences of the United States of America.

[64] Chang YW, Chen S, Tocheva EI,

Sogaard-Andersen L, et al. Correlated cryogenic photoactivated localization

Treuner-Lange A, Lobach S,

microscopy and cryo-electron tomography. Nature Methods.

[59] Raccuglia G. Gray platelet syndrome. A variety of qualitative platelet disorder. The American Journal

of Medicine. 1971;**51**(6):818-828

[60] Drouin A, Favier R, Masse JM, Debili N, Schmitt A, Elbim C, et al. Newly recognized cellular abnormalities in the gray platelet syndrome. Blood.

[61] Wang R, Stone RL, Kaelber JT, Rochat RH, Nick AM, Vijayan KV, et al. Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**(46):14266-14271

[52] Bentfeld-Barker ME, Bainton DF. Identification of primary lysosomes in human megakaryocytes and platelets.

[53] Boudreau LH, Duchez AC, Cloutier N, Soulet D, Martin N, Bollinger J, et al. Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation. Blood.

Blood. 1982;**59**(3):472-481

2014;**124**(14):2173-2183

[54] Marcoux G, Magron A, Sut C, Laroche A, Laradi S,

Hamzeh-Cognasse H, et al. Plateletderived extracellular vesicles convey mitochondrial DAMPs in platelet concentrates and their levels are associated with adverse reactions. Transfusion. 2019;**59**(7):2403-2414

[55] Heijnen HF, Oorschot V, Sixma JJ, Slot JW, James DE. Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from alphagranules to the cell surface. The Journal of Cell Biology. 1997;**138**(2):323-330

[56] Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, et al. Cryo-electron microscopy of vitrified specimens. Quarterly Reviews of Biophysics. 1988;**21**(2):129-228

[57] Koster AJ, Grimm R, Typke D, Hegerl R, Stoschek A, Walz J, et al. Perspectives of molecular and cellular electron tomography. Journal of

Structural Biology. 1997;**120**(3):276-308

**18**

[65] Wang Q, Mercogliano CP, Lowe J. A ferritin-based label for cellular electron cryotomography. Structure. 2011;**19**(2):147-154

[66] Mercogliano CP, DeRosier DJ. Concatenated metallothionein as a clonable gold label for electron microscopy. Journal of Structural Biology. 2007;**160**(1):70-82

[67] Beck JS, Berg BN. The circulatory pattern in the islands of Langerhans. The American Journal of Pathology. 1931;**7**(1):31-61

[68] Weigert R, Sramkova M, Parente L, Amornphimoltham P, Masedunskas A. Intravital microscopy: A novel tool to study cell biology in living animals. Histochemistry and Cell Biology. 2010;**133**(5):481-491

[69] Masedunskas A, Milberg O, Porat-Shliom N, Sramkova M, Wigand T, Amornphimoltham P, et al. Intravital microscopy: A practical guide on imaging intracellular structures in live animals. BioArchitecture. 2012;**2**(5):143-157

[70] Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nature Medicine. 2002;**8**(10):1175-1181

[71] Bellido-Martin L, Chen V, Jasuja R, Furie B, Furie BC. Imaging fibrin formation and platelet and endothelial cell activation in vivo. Thrombosis and Haemostasis. 2011;**105**(5):776-782

[72] Adili R, Holinstat M. Formation and resolution of Pial microvascular thrombosis in a mouse model of thrombotic thrombocytopenic Purpura. Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;**39**(9):1817-1830

[73] Malehmir M, Pfister D, Gallage S, Szydlowska M, Inverso D, Kotsiliti E,

et al. Platelet GPIbalpha is a mediator and potential interventional target for NASH and subsequent liver cancer. Nature Medicine. 2019;**25**(4):641-655

[74] Da Q, Derry PJ, Lam FW, Rumbaut RE. Fluorescent labeling of endogenous platelets for intravital microscopy: Effects on platelet function. Microcirculation. 2018;**25**(6):e12457

[75] Merrill-Skoloff G, Atkinson BT, Furie BC, Furie B. Real time in vivo imaging of platelets during thrombus formation. In: Michelson A, editor. Platelets. 3rd ed. Netherlands: Elsevier; 2013. pp. 635-652

[76] Lu Y, Li Q, Liu YY, Sun K, Fan JY, Wang CS, et al. Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases. Scientific Reports. 2015;**5**:13824

[77] Chen D, Uhl CB, Bryant SC, Krumwiede M, Barness RL, Olson MC, et al. Diagnostic laboratory standardization and validation of platelet transmission electron microscopy. Platelets. 2018;**29**(6):574-582

**21**

**Chapter 2**

**Abstract**

Receptors

physiological conditions.

infection

**1. Introduction**

Molecular Aspects of

Pathophysiology of Platelet

Receptor is a dynamic instrumental surface protein that helps to interact with specific molecules to respond accordingly. Platelet is the smallest in size among the blood components, but it plays many pivotal roles to maintain hemostasis involving its surface receptors. It (platelet) has cell adhesion receptors (e.g., integrins and glycoproteins), leucine-rich repeats receptors (e.g., TLRs, glycoprotein complex, and MMPs), selectins (e.g., CLEC, P-selectin, and CD), tetraspanins (e.g., CD and LAMP), transmembrane receptors (e.g., purinergic—P2Y and P2X1), prostaglandin receptors (e.g., TxA2, PGH2, and PGI2), immunoglobulin superfamily receptors (e.g., FcRγ and FcεR), etc. on its surface. The platelet receptors (e.g., glycoproteins, protease-activated receptors, and GPCRs) during platelet activation are over expressed and their granule contents are secreted (including neurotransmitters, cytokines, and chemokines) into circulation, which are found to be correlated with different physiological conditions. Interestingly, platelets promote metastasis through circulation protecting from cytolysis and endogenous immune surveillance involving several platelets receptors. The updated knowledge about different types of platelet receptors in all probable aspects, including their inter- and intra-signaling mechanisms, are discussed with respect to not only its (platelets) receptor type but also under different patho-

**Keywords:** platelet receptors, thrombus, cancer, aging, cardiovascular disease, viral

Platelets including other blood components were first time drawn by George Gulliver in 1841 (though it was not named platelet at that time) when he was working with a newly made compound microscope with twin lens [1]. Giulio Bizzozero at the end of the nineteenth century [2] first time coined the term and identified as platelets. Blood platelet in its form is found in mammals, but in birds and amphibians, it is present in circulation as intact mononuclear thrombocytes [3]. Platelets are not true cells, and it is classified as cell fragments (from megakaryocyte by the megakaryocytopoiesis) having no nucleus inside. Platelets circulate in the bloodstream and remain alive for 7–10 days. They (platelets) principally survey the inner lining of blood vessels. If they detect any breaches, they seal them in the vasculature by the creation of thrombi [4]. Platelets generally remain in inactive state

*Mrinal K. Poddar and Soumyabrata Banerjee*

## **Chapter 2**
