The Evolution of Mammalian Platelets

PART I

1

Platelet Biology

The Evolution of Mammalian Platelets Jack Levin Departments of Laboratory Medicine and Medicine, University of California School of Medicine, San Francisco, CA, United States

INTRODUCTION 1 INVERTEBRATES 1 NONMAMMALIAN VERTEBRATES 3 COMPARATIVE HEMOSTASIS 6 A COMPARISON OF HUMAN PLATELETS AND LIMULUS AMEBOCYTES 6 THE EVOLUTION OF HEMOSTASIS AND BLOOD COAGULATION 11 MEGAKARYOCYTES AND MAMMALS 13 Marine Mammals 15 Order Monotremata 15 Order Marsupialia 16 Platelet Levels 17 A Comparison of Nonplacental and Placental Mammals 18 CONCLUSIONS 18 ACKNOWLEDGMENTS 19 REFERENCES 20

INTRODUCTION The mammalian platelet is derived from the cytoplasm of megakaryocytes, the only polyploid hematopoietic cell. Polyploid megakaryocytes and their progeny, nonnucleated platelets, are found only in mammals. In all other animal species, cells involved in hemostasis and blood coagulation are nucleated. The nucleated cells primarily involved in nonmammalian, vertebrate hemostasis are designated thrombocytes to distinguish them from nonnucleated platelets. In many invertebrates, only one type of cell circulates in the blood (or hemolymph), and this single type of cell is typically involved in multiple defense mechanisms of the animal, including hemostasis. These cells are capable of aggregating and sealing wounds. This process is probably the earliest cellbased hemostatic function. A comparison of amebocytes (the only type of circulating cell in the hemolymph of the horseshoe crab, Limulus polyphemus) with human platelets provides a basis for understanding the many nonhemostatic functions of platelets.1–4 Platelets appear to possess many of the multiple capabilities that characterize “primitive” circulating amebocytes, only one of whose functions is hemostasis. For example, platelets possess rudimentary bactericidal and phagocytic activity (see Chapter 29). They have been shown to interact with bacteria, endotoxins, viruses, parasites, and fungi. Platelets not only are important for the maintenance of hemostasis but also are inflammatory cells (see Chapter 28). Despite these overall Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00001-1 Copyright © 2019 Elsevier Inc. All rights reserved.

similarities in function, there remains no proof that invertebrate blood cells evolved into platelets. Furthermore, the fact that nonnucleated platelets and their polyploid megakaryocyte progenitors in the bone marrow are present only in mammals suggests that some important feature of mammalian physiology benefits from this unique mechanism for the production of anucleate cells from the cytoplasm of a larger cell, for the apparently major purpose of supporting hemostasis. However, because both monotremes, which are egg-laying mammals, and marsupials, which have a nonplacental pregnancy, possess megakaryocytes and platelets, it is apparent that neither live birth nor the presence of a placenta accounts for the evolution of platelets in mammals. Therefore, the biological advantage gained from the generation of nonnucleated platelets by polyploid megakaryocytes remains unidentified (Figs. 1.1 and 1.2).

INVERTEBRATES In many marine invertebrates, only one type of cell circulates in the blood or is present in the coelomic fluid. This single cell type plays multiple roles in the defense mechanisms of the animal, including hemostasis. Such cells are capable of aggregating and sealing wounds. Although the biochemical basis for adhesion of these cells is not understood, the participation of cell aggregation in invertebrate hemostasis suggests that this process is perhaps the earliest cell-based hemostatic function (Figs. 1.1 and 1.2).1–4 The hemocytes of the ascidian Halocynthia roretzi, for example, aggregate after removal from the hemolymph (i.e., the circulating body fluid, equivalent to blood, in animals with open circulatory systems).3 Aggregation depends on divalent cations and is inhibited by ethylene diamine tetraacetic acid (EDTA). Similar to the response of mammalian platelets to vascular trauma, repeated sampling of hemolymph from the same area of an individual H. roretzi results in hemocytes that are increasingly activated, as measured by aggregometry (Fig. 1.3). Furthermore, the aggregated hemocytes release a factor into the plasma that induces additional aggregation. The hemocytes of the California mussel Mytilus californianus rapidly aggregate and adhere to foreign surfaces after the removal of blood from the animal.8 Aggregation in vitro was shown to be a two-step process and distinct from adhesion. Adhesion is Ca++- or Mg++-dependent.8 The parallels with mammalian platelet function are evident. The enormous range of types of cell-based coagulation in insects has been described by Gr
egoire.9 The hemolymph of many insects contains coagulocytes, which on contact with a foreign surface extrude long, straight, threadlike processes that may contain cytoplasmic granules. These cytoplasmic extensions mesh with similar processes from other coagulocytes, creating a hemostatic plug. Examples of coagulocyte aggregation and cytoplasmic expansions are shown in Figs. 1.4 and 1.5. Thrombocytoids, a type of circulating hemocyte in the

1

2

PART I Platelet Biology

Fig. 1.1 Aggregation and alteration in the shape of Limulus amebocytes during cellular clotting on a glass surface. Note the prominent nucleus and the variety of shapes that occur after activation. The cells have also become degranulated (see also Figs. 1.20, 1.22, and 1.23). Magnification
200 (upper) and
320 (lower). (From Levin and Bang,1 with permission.)

hemolymph of the blowfly Calliphora erythrocephala tend to fragment, resulting in “anucleated cytoplasmic fragments” (Fig. 1.6).10,11 Their fragments then aggregate and form networks which are believed to promote hemostasis and seal wounds.10,11 Each cell fragment remains surrounded by an intact plasma membrane. The authors conclude that “The thrombocytoid of this insect in some respects resembles the megakaryocyte of mammals. The transformations exhibited by these cells..., such as increased fragmentation and agglutination, are analogous to the process of agglutination of blood platelets in mammals giving rise to the white thrombus.” In some invertebrates, hemostasis is provided entirely by cell aggregation at the site of a wound.12–14 In others, the hemocytes contain coagulation factors or clottable proteins that are

released after activation and/or aggregation of the cells.15–18 In the Arthropoda, as in other invertebrate classes, mechanisms of hemostasis vary widely. In decapod crustaceans (e.g., Homarus americanus, the American lobster), hyaline cells, a type of hemocyte, initiate coagulation when they lyse.19 In some arthropods, circulating amebocytes release a coagulase that activates a clottable protein already in the plasma.20 Plasma from the hemolymph of L. polyphemus, the American horseshoe crab, normally lacks coagulation factors.16,17 However, after activation, amebocytes, the only type of circulating blood cell in Limulus, release a cascade of coagulation factors that result in coagulation of the plasma.21,22 Activation of Limulus amebocytes not only results in initiation of coagulation, but also is associated with the initiation of other defense mechanisms, including

The Evolution of Mammalian Platelets

3

1

Fig. 1.3 Activation of hemocyte aggregation in Halocynthia roretzi. Point A represents the point on the tunic of the animal through which the hemolymph was repeatedly taken; point B represents a different point on the tunic. The time between the initial collection of hemolymph (0min) and subsequent collections is indicated above each tracing. The bar represents 5 min. The extent of light scattering is shown on the ordinate in arbitrary units. (From Takahashi et al.,3 with permission.)

Fig. 1.2 Appearance of long filamentous processes after the activation of Limulus amebocytes. These processes are often connected with those of other amebocytes. Magnification
200 (upper) and
320 (middle and lower). (From Levin and Bang,1 with permission.)

wound healing, activation of a primitive complement system, and release of antibacterial factors.23

NONMAMMALIAN VERTEBRATES Nonmammalian vertebrates have nucleated, often spindleshaped thrombocytes, the first cells to evolve that specialize in hemostasis.12,15,24 Thrombocytes are found in fish, and in some species multiple types of thrombocytes have been described.25,26 However, some reports of multiple types of thrombocytes may have inadvertently described technical artifacts of the methods of blood collection, which resulted in cell activation and morphological alterations of some of the thrombocytes. Thrombocytes have been variously described as spindle shaped (fusiform), spiked, spherical, oval, or teardrop in appearance, with a few but variable number of cytoplasmic granules.27 There are a central round or elongated nucleus and a rim of cytoplasm. The nuclear: cytoplasmic ratio is greater than that of the nucleated red blood cells. Periodic acid-Schiff (PAS)-positive cytoplasmic granules are sometimes described.

Fig. 1.4 Dytiscus marginalis L. (Coleoptera). Clustering of coagulocytes around a fragment of cuticle stimulates the reaction of hemolymph at wound sites. Magnification
800. (From Gr
egoire,9 with permission.)

In addition, size differences may exist between immature and mature thrombocytes.28 Quantification of fish thrombocytes has been made difficult by the often-described resemblance between fish thrombocytes and lymphocytes, and the tendency of thrombocytes to clump.25 The most extensive quantitative

4

PART I Platelet Biology

Fig. 1.5 Erodius tibialis (Coleoptera). The threadlike cytoplasmic processes are shown, carrying along the granules produced by six coagulocytes (asterisks).
goire,9 with Magnification
960. (From Gre permission.)

Fig. 1.6 Ultrathin section of a thrombocytoid with numerous deep invaginations. The cytoplasm is described as demonstrating rough surfaced endoplasmic reticulum with cysternae whose profiles are parallel and characteristically curved. Orig. magnification
12,000. (From Zachary et al.,10,11 with permission.)

study, based on Wright-stained blood smears, has reported that 52% (mean; range, 46%–61%) of white blood cells in 121 species of marine fishes are thrombocytes.29 In that study, thrombocytes were overall the most common type of white blood cell. Cytochemical analysis has been successful in distinguishing thrombocytes from lymphocytes in fish.30,31 Thrombocytes of the piracanjuba Brycon orbignyanus30 and the Murray cod Maccullochella peelii peelii31 were PAS-positive, but negative for a wide range of other cytochemical markers (Fig. 1.7). In contrast, the thrombocytes of seven other species of fish were positive for both PAS and acid phosphatase.32,33 The thrombocytes of the relatively primitive Australian lungfish Neoceratodus forsteri contain, in addition to PAS, variable amounts of acid phosphatase, gamma-naphthyl acetate esterase (ANAE), and AS-D chloroacetate esterase (AS-D).34 Examples of thrombocytes in cartilaginous fish are shown in Fig. 1.8.35 A single von Willebrand factor transcript that encodes a simpler protein compared with higher vertebrates has been identified in the jawless Atlantic hagfish, Myxine glutinosa (Fig. 1.9).36 It is present in both thrombocytes and endothelial cells. Some species of estuarine cyprinodontiform fishes have been described as having a seasonal variation in the ratio of mature to immature circulating thrombocytes.28 Immature thrombocytes reached their highest levels during July and August, which the authors interpreted as indicating an increased rate of thrombopoiesis. Fish thrombocytes contain bands of microtubules33,37 and in at least some species are described as phagocytic.33,38–40 In contrast to platelets, thrombocytes are not typically aggregated by adenosine diphosphate (ADP), or epinephrine.12,41,42 Zebrafish (Danio rerio) thrombocytes have been shown in vivo to play a role in the development of arterial thrombi.43 However, avian thrombocytes

demonstrated a much lesser ability to occlude an experimentally damaged carotid artery than did murine platelets, perhaps because the density of αIIbβ3 receptors on human platelets is at least 18–25 times greater than on chicken thrombocytes.44 Avian thrombocytes are similar in appearance to the thrombocytes in fish and are believed to be produced by mononuclear precursors in the bone marrow.45 Four developmental stages of thrombocyte precursors have been described in the chick embryo, and collectively account for 0.6–2.4% of nucleated cells in the bone marrow.46 PAS-positive cytoplasmic granules were present in all stages of the thrombocytic lineage. An extensive ultrastructural study of six species of domestic birds concluded that although thrombocytes are similar in size to lymphocytes, thrombocytes have a denser nucleus and a very highly vacuolated cytoplasm.47 Flow cytometry has been used to discriminate between lymphocytes and thrombocytes in chickens48 and ducks.49 The developmental stages of the thrombocyte lineage in chicken are shown in Fig. 1.10.50 Phagocytosis has been demonstrated.51 As in fish, thrombocytes are the most common white blood cell in the chicken48 and ducks.49 Similar to platelets, avian thrombocytes contain serotonin (5-hydroxytryptamine)52,53 and release what appears to be β-thromboglobulin during the release reaction.54 The thrombocytes of at least some birds, amphibians, reptiles, and fish have a membrane system referred to as the surface-connected canalicular system (SCCS).37,55–59 This system is also a feature of mammalian platelets and has been linked to their derivation from the cytoplasm of megakaryocytes.60 However, the presence of the SCCS in nonmammalian thrombocytes indicates that this system reflects an important function of blood cells that play a major role in hemostasis, and that the SCCS need not be derived from the demarcation membrane system (DMS) of megakaryocyte cytoplasm. Bovine61 and African elephant (Loxodonta africana)62 platelets are apparently exceptions in that they do not contain a SCCS, although bovine megakaryocytes have a DMS.60 Importantly, the platelets of the African elephant also lack microtubules.62 Elegant studies of the thrombocytes of the dogfish Mustelus canis demonstrated that the series of cytoskeletal changes that occurred following exposure to thrombin paralleled those of platelets.63 Furthermore, the authors concluded that the responsiveness of nucleated fish thrombocytes to mammalian thrombin indicated the presence of evolutionarily conserved signal transduction pathways. Presumably, this conclusion can be applied to the thrombocytes of other nonmammalian vertebrates. Thrombocytes from an alligator (a reptile) and a bullfrog (an amphibian) are shown in Fig. 1.11. Multiple examples of the thrombocytes of other nonmammalian vertebrate species are shown in Fig. 1.12.64–68 Cytochemical studies of the thrombocytes of the giant lizard Gallotia simonyi (a reptile) failed to detect any of the substrates for the six cytochemical stains utilized, including PAS and four enzymes.69 However, the thrombocytes of two species of Australian crocodiles were PAS-positive.70 Serotonin has been detected immunochemically in seagull, alligator, and sea turtle thrombocytes

The Evolution of Mammalian Platelets

ACP

ALP

b-glu

Thrombocyte

Lymphocyte

Peroxidase

5

NAE

SBB

PAS

NBE

Thrombocyte

Lymphocyte

Thrombocyte

Lymphocyte

NCE

Fig. 1.7 Murray cod (Maccullochella peelii peelii) lymphocytes and thrombocytes. Light microscopy of blood smears. Top: enzymatic staining for nonesterases. Peroxidase (A,B); acid phosphatase (ACP) (C,D); alkaline phosphatase (ALP) (E,F); and β-glucuronidase (β-glu) (G,H). Middle: enzymatic staining for esterases. Naphthol AS chloroacetate esterase (NCE) (A,B); naphthyl acetate esterase (NAE) (C,D); and α-naphthyl butyrate esterase (NBE) (E,F). Bottom: nonenzymatic staining: Sudan black B (SBB) (A,B); and periodic acid Schiff (PAS) (C,D). In contrast to lymphocytes, thrombocytes were positive only for PAS, which indicated the presence of glycogen (bottom panel, D). Magnification
100. Bar ¼ 10 μm. (Modified from Shigdar et al.,31 with permission.)

(Fig. 1.13) and its presence in alligator and sea turtle thrombocytes confirmed with HPLC (Fig. 1.14).53 Serotonin has not been detected in the thrombocytes of the American bullfrog Rana catesbeiana53 or the tortoise Geoclemys reevesii.71 Phylogenetic insights have been gained from an analysis of the presence of serotonin in the thrombocytes and platelets of vertebrates and selected mammalian orders, respectively (Fig. 1.15).53 Studies of the African clawed frog, Xenopus laevis, using an antibody against thrombocytes, identified cells in the liver and spleen that expressed CD41 and had higher ploidy levels than the nucleated, tetraploid (4 N) erythrocytes.72 Hepatic thrombocytes had higher DNA levels that did circulating

thrombocytes (Fig. 1.16).72 Circulating thrombocytes had a mean ploidy level of 8 N and were acetylcholinesterase positive, in contrast to erythrocytes. When the hepatic cells of X. laevis were cultured in the presence of recombinant X. laevis thrombopoietin (TPO), the cells differentiated into polyploid CD41expressing cells73 (Fig. 1.17). The subsequent culture of these cells after removal of TPO resulted in the production of mature, spindle shaped thrombocytes, which were activated by thrombin (Fig. 1.18). Therefore, these cells were considered to have features similar to mammalian megakaryocytes. It will be instructive to determine the ploidy levels of the precursors of thrombocytes in other nonmammalian vertebrates.

1

6

PART I Platelet Biology

Fig. 1.8 Transmission electron micrographs of thrombocytes in cartilaginous fish (Chondrichthyes). Left: dogfish (Squalus acanthias) thrombocyte. Note the single nucleus in the lower region of the cell. A group of peripheral microtubules, sectioned lengthwise, are present in the upper left part of the cell. Right: skate (Raja eglanteria) thrombocytes after their incubation with skate thrombin. Four thrombocytes have aggregated, with a loss of distinct cytoplasmic boundaries. The aggregation and fusion of nucleated thrombocytes after exposure to thrombin resembles the response of human platelets to thrombin. In contrast to platelets, adenosine diphosphate does not cause aggregation of thrombocytes. (From Lewis,34 with permission).

Fig. 1.9 Hagfish (Myxine glutinosa) von Willebrand Factor (VWF) is expressed in peripheral blood. Fluorescence microscopy images of hagfish blood processed for immunofluorescence staining of VWF, using polyclonal antihuman VWF antibody. Left image (i) shows 4 VWF-positive cells (arrows), which are smaller than neighboring VWF-negative erythrocytes (One of these is outlined in white). Right image (ii) is a higher power view showing the punctate staining pattern of a VWF-positive cell surrounded by VWF-negative erythrocytes and spindle cells. (From Grant et al.,36 with permission.)

COMPARATIVE HEMOSTASIS An overview of comparative hemostasis is provided in Table 1.1.74 Because of the great heterogeneity in types of blood cells and coagulation mechanisms in invertebrates, any attempt to summarize the characteristics of hemostasis in these animals cannot avoid oversimplification and inaccuracy. Overall, however, it is clear that when cells are present in the invertebrate circulation or coelomic fluid, they always play a role in hemostasis.12,75 Among the extensive original studies of the blood by William Hewson (1739–1774) is a highly instructive plate that illustrates the “red particles of the blood” in a wide variety of animals76 (Fig. 1.19). The following statements in Hewson’s text accompany this plate: In the blood of some insects the vesicles [blood cells] are not red, but white, as may easily be observed in a lobster (which Linnaeus calls an insect), one of whose legs being cut off, a quantity of a clear sanies flows from it; this after being some time exposed to the air jellies, but less firmly than the blood of more perfect animals. When it is jellied it is found to have several white filaments; these are principally the vesicles concreted, as I am persuaded from the

following experiment. ... There is a curious change produced in their shape by being exposed to the air, for soon after they are received on the glass they are corrugated, or from a flat shape are changed into irregular spheres. This change takes place so rapidly, that it requires great expedition to apply them to the microscope soon enough to observe it.76 (pp. 233–234). Hewson (and his colleague Magnus Falconar) were actually observing the hemocytes of lobsters becoming activated after removal from the animal, changing shape, and then aggregating. Note the obviously altered appearance of the hemocytes, in contrast to the multiple examples of genuine “red particles of the blood” (in Fig. 1.19, an original Hewson plate; compare his Figs. 11 and 12).

A COMPARISON OF HUMAN PLATELETS AND LIMULUS AMEBOCYTES L. polyphemus, the horseshoe crab, is the last surviving member of the class merostomata, which included marine spiders. The Limulus amebocyte, which is the only type of circulating blood cell in the hemolymph, has probably been the most intensely

The Evolution of Mammalian Platelets

7

1

357

358

359

360

361

362

363

364

365

Fig. 1.10 Camera lucida drawings of the stages of maturation of the thrombocyte lineage in the bone marrow of the white leghorn chicken. Thromboblasts (357, 358); early immature thrombocytes (359–362); midimmature thrombocyte (363); late immature thrombocyte (364); mature thrombocyte (365). (From Lucas and Jamroz,50 with permission.)

Fig. 1.11 Transmission electron micrographs of thrombocytes. Left: alligator (Alligator mississippiensis) thrombocytes. The three thrombocytes demonstrate a spindle shape, large nuclei, and a fine open canalicular system. Right: bullfrog (R. catesbeiana) thrombocytes. Two of the thrombocytes contain large nuclei. The inclusion in the lowest cell may be a phagocytosed red blood cell. (From Lewis,35 with permission.)

studied of the invertebrate blood cells involved in hemostasis and blood coagulation.1,77 The concentration of amebocytes in the blood of adult limuli is approximately 15
109/L. Amebocytes are nucleated cells that are approximately the size of mammalian monocytes. Their cytoplasm is packed with

granules (Fig. 1.20).5 After activation on a foreign surface (or by bacterial endotoxins), amebocytes spread and degranulate (Fig. 1.20, right panel). Degranulation is associated with exocytosis.16,78–80 The amebocytes, which are normally discoid (like platelets), develop pseudopodia and microspikes.81 These cells

8

PART I Platelet Biology

Fig. 1.12 Thrombocytes of the common lizard (Podarcis S. sicula Raf.), dogfish (Scyliorhynus stellaris L.), electric ray (Torpedo marmorata), red-legged partridge (Alectoris rufa rufa L.), eagle (Aquila rapax), and Brujn’s echidna (Zaglossus bruijni). Top panel: A. €nwald Giemsa). B. Lizard thrombocytes positively stained for acid phosphatase. C. Dogfish thrombocyte with group of lizard thrombocytes (May Gru granules stained for β-glucuronidase. Lower left panel, A–C: A. torpedo thrombocyte (T) positively stained and basophilic erythroblast (Eb) negatively stained for platelet factor 4 (PF4). [Inset: Thrombocyte (left) positive and eosinophilic granulocyte (E) negative for PF4]. B. Red-legged partridge thrombocytes positively stained for α-naphthylacetate esterase. C. Red-legged partridge thrombocytes positively stained for acid phosphatase. Lower right panel, D: Eagle thrombocytes (T), in a field that contains a heterophile (right), basophile (left), and many nucleated red blood cells. In this figure, the relative sizes of the thrombocytes in the species shown are not to scale. Note the tendency of thrombocytes to clump (top panel: A and B; lower left panel: B and C). The cleft that sometimes appears in the nucleus of thrombocytes has been described in many reports (top panel: C). Lowest right panel, F: variably shaped thrombocytes in Bruijn’s echidna. (From Pica et al.,64–66 D’Ippolito et al.,67 and Jain,68 with permission.)

are also capable of retraction (Fig. 1.21),82 apparently similar to the process of clot retraction induced by mammalian platelets. Amebocytes have a Toll-like receptor and have been shown to bind bacterial endotoxin.83,84 These cells are also capable of phagocytosis.85 Fig. 1.22 6 shows the ultrastructure of a Limulus amebocyte. The cytoplasm contains at least two types of granules (Fig. 1.23)7 that are biochemically different and contain all the components of the blood coagulation mechanism.7,21,77 Marginal microtubule bands are present (Fig. 1.24).

Mammalian platelets are appropriately considered as functioning primarily to support a range of hemostatic mechanisms, both by maintaining the integrity of blood vessels and by contributing to the process of blood coagulation. However, platelets have many other capabilities, although often rudimentary, that appear to be unrelated to their hemostatic function. Platelets have been shown to be bactericidal for B. subtilis and E. coli (but not S. aureus), but only in the presence of thrombin and a heat-labile plasma protein fraction >100 kDa.86 The bactericidal mechanism has not been not

The Evolution of Mammalian Platelets

Fig. 1.13 Immunocytochemical evidence for serotonin in human platelets and in seagull and alligator thrombocytes. Platelets or thrombocytes were incubated with a specific serotonin antibody and a fluorescently labeled secondary antibody. The morphology of human platelets (A), seagull thrombocytes (C), and alligator thrombocytes (E) is shown. Leukocytes and erythrocytes are also present in (C) and (E). The bright spots indicate the presence of serotonin-specific fluorescent labeling in granules in platelets (B), and in seagull (D) and alligator (F) thrombocytes. Bar ¼ 10 μm. (From Maurer-Spurej,53 with permission.)

1000

Human

Voltage (mV)

800

Alligator 600

Sea turtle 400 200

Fresh water turtle, frog, tuna, salmon

0 4

6

8

10

12

14

16

Time (min) Fig. 1.14 Quantitative determination of serotonin with HPLC. Serotonin levels in isolated platelets or thrombocytes were determined chromatographically. Human platelets and alligator and sea turtle thrombocytes contain serotonin, but the thrombocytes of the fresh water turtle, frog, tuna, or salmon do not. (From Maurer-Spurej,53 with permission.)

9

established. Despite approximately 450 million years of evolution,87 mammalian platelets retain many of the functions of Limulus amebocytes (and of many other comparable invertebrate blood cells). This phenomenon may have resulted from the retention of multiple functions previously found in a single, “all-purpose” circulating cell type, such as the Limulus amebocyte, only one of whose functions was hemostasis. Studies of the thrombocytes of the rainbow trout Oncorhynchus mykiss concluded that these cells had both hemostatic and phagocytic functions. The authors speculated that “some aspects of this dual functionality observed in thrombocytes may have been lost with the evolution of the anucleate platelet.”88 The multiple characteristics shared by mammalian platelets and Limulus amebocytes are summarized in Table 1.2. Platelets have rudimentary bactericidal and some phagocytic or phagocytic-like activity.86,90–93 However, an ultrastructural study of interactions between human platelets and various-size particles concluded that platelets were not true phagocytes, and that the uptake of bacteria involved the channels of the open canalicular system (OCS).94 Nevertheless, an exhaustive review of studies that described the interactions of thrombocytes and platelets with particulate materials or bacteria presented considerable data that appeared to support the conclusion that both thrombocytes and platelets are capable of phagocytosis.95 Platelets contain endotoxin-binding substances96 and have been shown to interact with bacteria, endotoxins, viruses, and fungi.97–101 Murine102 and human103 platelets and chicken thrombocytes104 have been shown to express a functional Toll-like receptor-4 (TLR4), the receptor for bacterial endotoxin (LPS).102 The extensive role of platelets in antimicrobial host defense101,105 is reviewed in Chapter 29. Staphylococcus can stimulate human platelets to undergo the release reaction.106 Microbicidal proteins polymorphic protein-1 (PMP-1) and PMP-2 are small cationic peptides released by rabbit platelets, which disrupt the membrane of S. aureus and cause cell death.107 It has been demonstrated that staphylococci have a receptor for the Fc fragment of immunoglobulin G (IgG) that provides a mechanism for aggregation of human platelets by the formation of a complex composed of bacteria, IgG, and platelets.108 Furthermore, it has been shown that platelet binding by S. aureus is mediated, at least in part, by direct binding of clumping factor A (ClfA) to a novel 118-kDa platelet membrane receptor.109 Pneumococcus (S. pneumoniae) and vaccinia virus can induce the release of serotonin from human platelets.110,111 Platelets may preferentially carry oral streptococci to damaged endocardium due to their serine-rich repeat adhesins, which target platelet sialoglycans.112 Fibrinogen in conjunction with other unidentified plasma factors was required for platelet aggregation and secretion.113 The aggregation of human platelets by S. viridans, S. pyogenes, and S. sanguis has been demonstrated.114–116 Human platelets have been reported to be cytotoxic for parasites by a mechanism involving an IgE receptor on the platelet surface.117,118 Platelets also have been shown to play a role in the excretion of Schistosoma mansoni in the stool of mice.119 Other studies have demonstrated that platelets enhance the adherence of schistosome eggs to endothelial cells, and that interleukin (IL)-6, produced by activated monocytes, markedly increases the cytotoxicity of platelets against schistosomula.120 Under certain circumstances, platelets demonstrate a chemotactic response and migrate,121–123 as do amebocytes.124 It has been concluded that the thrombocytes of the turbot Psetta maxima are also capable of movement.33 Recent studies have convincingly documented this capability of platelets by demonstrating that they are able to function as cellular scavengers, and collect bacteria from the vascular surface.125 The data indicate that platelets can function as mechano-sensors and interacted importantly with neutrophils and the immune system. Direct involvement of αIIbβ3 integrin in platelet migration

1

10

PART I Platelet Biology

4000 3000 2000 1000 900 800

700

600

500 400 300 200 100

90

80

70

60

50

40

30

20

10

1

Million years ago Fossil time Humans

Circulating serotonin present

Gorillas Monkeys Cats, dogs

Pigs

310

Cattle Birds Living reptiles Fishes Vertebrates Protists Origin of eukaryotes Prokaryotes Fig. 1.15 Phylogenetic comparison of species with and without circulating serotonin. Fossil records were used for the comparison of species divergence times. Time estimates were based on previously published studies. The time scale is linear only within the ranges 10–100, 100–1000, and 1000–5000 million years. Serotonin appears in the circulation in the American alligator and leatherback turtle, reptiles that evolved approximately 310 million years ago. Avian and mammalian blood also contains serotonin. (From Maurer-Spurej,53 with permission.)

4N

T12+ T12−

4N

Spleen

T12+ T12−

Count

Count

PB

0

0 101

102

103

104

105

101

DNA contents

102

103

104

105

DNA contents

4N

Count

Count

Fig. 1.16 DNA content analysis of polyploid T12+ cells (African clawed frog, Xenopus laevis thrombocytes) in the peripheral blood, spleen, and liver. (A–C) Each panel shows the DNA histograms of cells labeled with T12 and Hoechst 33342; T12+ (black line) and T12 (gray fill) DNA histograms. (D) White, black, and gray areas of the histogram indicate peripheral blood, spleen, and liver T12+ cells, respectively. (From Tanizaki et al.,72 with permission.)

PB T12+ Spleen T12+ Liver T12+

T12+ T12−

Liver

0

0 101

102

103

104

DNA contents

105

101

102

103

104

DNA contents

105

11

The Evolution of Mammalian Platelets

was demonstrated.125 Collectively, these and other observations indicate that bacteria, viruses, fungi, and parasites are capable of interacting with platelets. Depending upon the nature of the infectious particles and other poorly understood variables, this interaction can result in platelet aggregation, release of platelet constituents, phagocytosis of the infectious agent, and ultimately a shortened platelet life span. (See Chapter 29 for further details.)

THE EVOLUTION OF HEMOSTASIS AND BLOOD COAGULATION None of the previously described functions seems necessary for the current role of platelets in mammalian hemostasis. Therefore, it is likely that some of the capabilities of mammalian platelets are vestiges of functions originally present in the more “primitive” yet multicompetent cells from which mammalian blood cells have evolved. The roles of the amebocyte in providing hemostasis and controlling infection, and the reaction of the amebocyte to endotoxin, suggest that in various mammals the response of platelets and the blood coagulation system to Gram-negative infections or endotoxins is an evolutionary remnant of ancient mechanisms (Table 1.2). The limited ability of mammalian platelets to phagocytose (or internalize) particles

Fig. 1.17 Transmission electron micrograph of T12+ large cell on Day 8 of culture of hepatic cells from the African clawed frog, Xenopus laevis in the presence of recombinant TPO. Structures similar to the surface-connecting canalicular system and dense granules of megakaryocytes were observed. (From Tanizaki et al.,73 with permission.)

Fig. 1.18 (A) Xenopus laevis megakaryocyte-like cells following culture of hepatic cells. (B) Enriched megakaryocyte-like cells were cultured in the presence or absence of recombinant TPO. After 2 days in suspension culture in the absence of TPO, spindle shaped thrombocytes were observed (solid arrowheads). (From Tanizaki et al.,73 with permission.)

TABLE 1.1 Summary of Hemostatic Mechanisms Identified So Far in Various Groups of Animals

Thrombocytes/platelets Adhesion Aggregation Retraction Viscous metamorphosis Coagulation factor Vessel contraction Plasma coagulation Fibrinogen ! fibrina Prothrombin ! thrombin Spontaneous fibrinolysis a

Invertebrates

Cyclostomes

Elasmobranchs

Bony fish

Amphibians

Reptiles

Birds

Mammals

“+” + + + “+” + + + “+”

+

+

+

+

+

+ +

+

+

+

+ + +

+

+

+

+ + + 0

+ + + +

+ + + 0

+ + + + + + + + + + +

+ + + + + + + + + + +

0

In some examples, the term clottable protein would be more appropriate. Adapted from Hawkey.74 See also Needham.13

+ + + 0

+ + + + +

1

12

PART I Platelet Biology

entrapped within the extracellular blood clot of one of its target organisms, the Pacific white shrimp, Litopenaeus vannamei.126 Pertinently, it has been shown that both human and Limulus clots bind bacterial endotoxin (Fig. 1.25).127 An example of the association between hemostatic and antibacterial functions in platelets is demonstrated by the observation that S. aureus induces platelet aggregation through a fibrinogen-dependent mechanism.128 Quick129 wrote: “Even in man one does not only see residua of the more elementary hemostatic reactions, but actually these phylogenetically older mechanisms still function effectively, but in a restricted manner” (p. 2). And Laki,130 referring to the evolution of blood coagulation and hemostasis, stated: “... man, who must realize that the imprints of a very distant past are still with him” (p. 305). Insightfully, Quick129 also considered “the possibility that the clotting mechanism may not have had as its primary purpose, hemostasis, but rather defense against bacterial invasion and repair of tissue” (p. 5). Needham13 has articulated the same hypothesis. In summary, platelets not only are important for the maintenance of hemostasis, but also are inflammatory cells (see Chapter 28) that have important roles in antimicrobial host defense mechanisms101,105,131,132 (see Chapter 29). Although the similarities in functions of platelets and amebocytes are consistent with the evolution of plasmatic blood coagulation in mammals from initially cell-based mechanisms in invertebrates, there is no proof of such an evolutionary trail, despite the parallel cellular functions and the similarity of the enzymatic components. Examples are the serine proteases upon which Limulus blood coagulation is based and some mammalian blood coagulation factors that are also serine proteases. Nevertheless, as eloquently stated by Quick129: The appearance in blood of a soluble protein that could be gelated appears a priori as another abrupt imposition of a new hemostatic process, but it is likely that such is not the case. It is probable that this new material in the plasma represents a transfer of an intracellular constituent to an extracellular state. If one looks upon the platelet as the descendant of a primitive coalescing cell, then one can understand why it has the power to aggregate and why it still contains fibrinogen. (p. 2).

Fig. 1.19 “A comparative view of the flat vesicles of the blood in different animals, exhibiting their size and shape.” The size of the red blood cell in man (Fig. 2) is compared with that in 23 animals grouped according to the similar size of their red blood cells, thus yielding the 12 figures (Figs. 1–12) in this plate. Figs. 11 and 12 show the hemocytes of the lobster before and after the shape change produced by a foreign surface. Note that these are the only cells observed among the 24 animals studied that changed shape and aggregated after removal from the animal (see text). Fig. 13 demonstrates “milk globules.” (From Hewson76 (1846), Plate V, p. 312 from Section III (originally published in 1777); from the author’s collection.)

and kill bacteria may be another remnant of functions that are more important in amebocytes (and the hemostatic cells of other invertebrates). Thus, these two cell types, amebocytes from an ancient marine invertebrate and platelets from mammals, have remarkably similar characteristics. The relative importance of their functions has changed with evolution, but after hundreds of millions of years, coagulation and antibacterial mechanisms remain at least partly linked. A pathogenic Gram-negative bacterium, Vibrio harveyi can be

Some evolutionary aspects of blood coagulation have been reviewed.12,20,21,39,133 Strikingly, basal chordates (protochordates) do not appear to have a plasma-based coagulation mechanism.133 Hemostasis in these primitive prevertebrates is dependent upon the aggregation of circulating cells at wound sites. In addition, the apparently sudden evolutionary appearance of nonnucleated platelets and megakaryocytes in mammals must be considered. This seems to be a marked departure from all other groups of animals, even taking into account the evolutionary concept of punctuated equilibria.134,135 However, as thoughtfully stated by Ratcliffe and Millar,136 “attempts to trace the origin of vertebrate blood cells within the invertebrates will at best be speculative and based on the assumption that comparisons with living species are valid since the enigmatic ancestral forms are no longer available” (p. 2). These authors also wrote: “It is most likely that these cells (i.e., invertebrate thrombocyte-like cells) are analogous rather than homologous to vertebrate platelets. They (i.e., platelets) may have arisen by convergent evolution due to similar evolutionary/environmental pressures” (p. 12). Intriguingly, it has been shown that the surface of avian thrombocytes presents analogues of human platelet GPIIb and GPIIIa, which are recognized by both polyclonal antibodies specific for the human GPIIb and GPIIIa subunits, and monoclonal anti-GPIIb-IIIa complex-specific antibodies.137–139 These studies have been supported by the observation that the aggregation of pigeon

The Evolution of Mammalian Platelets

13

1

Fig. 1.20 Limulus amebocytes. Three intact normal cells are shown on the left. The cytoplasm is packed with granules. Flattened, spread, degranulated amebocytes, after exposure to a foreign surface or a bacterial endotoxin, are shown on the right. Differential interference phase microscopy does not reveal the single large nucleus, which is located in the apparently depressed area in the middle of two of the cells in the left panel. Original magnification
1000. (From Levin,5 with permission.)

Fig. 1.21 Amebocyte tissue can be prepared for study by collecting blood under aseptic and endotoxin-free conditions in embryo watchglasses. After removal from Limulus, the blood cells settle and aggregate into a tissuelike mass that, after an extended period in vitro, undergoes contraction. In the right-hand watchglass, the mass is 1 day old. In the upper left of this watchglass, the amebocyte tissue mass has contracted into the compact, white, buttonlike mass. The fluid medium was Limulus plasma. (From €derha €ll et al.,82 with permission.) So

Fig. 1.22 Longitudinal section of a Limulus amebocyte. In this transmission electron micrograph, the cell is spindle shaped. A longitudinal section cut at right angles to this one would reveal a more oval shape. Large, homogeneous secretory granules are present. Magnification
7000. (From Copeland and Levin,6 with permission.)

thrombocytes by thrombin in the presence of calcium and fibrinogen was inhibited by an anti-GPIIb antibody.140 GPIIb-IIIa (integrin αIIbβ3) has also been detected on thromboblasts in chick embryos and importantly, the level of its

expression was related to the differentiation of thromboblasts (which were colony forming cells) into thrombocytes (not colony forming).139 The previously mentioned studies have been supported by the application of quantitative PCR and microarray analysis, which detected genes that encoded for the Mpl receptor, the α2 and β3 integrins, and the GPIb-V-IX receptor complex on chicken thrombocytes.44 Analogues of the GPIIb-GPIIIa complex have also been detected on the thrombocytes of the channel and related blue catfish, but not on the thrombocytes of seven other bony fish.141 Zebrafish thrombocytes also have been demonstrated to have platelet GPIb and GPIIb-IIIa-like complexes on their surfaces.142 In addition, Zebrafish thrombocyte precursors are c-mpl positive, and injection of an antisense c-mpl morpholino into embryos eliminated the production of thrombocytes.143

MEGAKARYOCYTES AND MAMMALS Nonnucleated platelets, and presumably their polyploid megakaryocyte progenitors in the bone marrow (and in the spleen in some animals), are present only in mammals. This suggests that some important feature of mammalian physiology benefits from this unique mechanism for producing an unprecedented

14

PART I Platelet Biology

Fig. 1.23 Left: Limulus amebocyte showing both major (asterisks) and minor (arrows) granules. Note the marked density of the smaller class of granules. In this section, the large nucleus is not present. Magnification
6140. Right: Scanning electron micrograph of a preparation of intact cytoplasmic granules obtained from Limulus amebocytes. Magnification
10,000. (Left: From Copeland and €rer et al.,7 with permission.) Levin,6 with permission; Right: From Mu

TABLE 1.2 Comparison of Limulus Amebocytes and Mammalian Plateletsa Characteristic or Function

Limulus Amebocyte

Mammalian Platelet

Hemostasis Blood coagulation Clottable protein Nucleus Viscous metamorphosis Cellular processes Granules Response to endotoxin Phagocytosis Antibacterial function Motility

Essential Essential Yes Yes Yes (?) Yes Yes Yes Yes Yes Yes

Essential Ancillary Yes No Yes Yes Yes Yes Yes (?) Yes Yes

a

Fig. 1.24 A marginal microtubule band is demonstrated in a crosssection of a Limulus amebocyte. Projections can be seen leading from one microtubule to another (arrows). These projections may serve to stabilize the microtubule band and are likely related to microtubuleassociated proteins present in other systems. Magnification
104,000. (From Tablin and Levin,81 with permission.)

anucleate cell from the cytoplasm of a larger cell, for the apparently major purpose of supporting hemostasis. However, because platelets have the rudimentary capacity to perform some of the functions that are carried out by other blood cell types in mammals, and because there is increased recognition that they also play a role in nonhemostatic defense mechanisms (Chapters 28 and 29), we must be cautious about assuming that hemostasis is the only major platelet function. In addition, all nonmammalian animal species require a mechanism to prevent hemorrhage or uncontrolled loss of body

From Levin, J. 1985.89 In: Blood Cells of Marine Invertebrates. Experimental systems in cell biology and comparative physiology. Cohen WD, editor. New York: Alan R. Liss, Inc.. p. 145–63.

fluids, and most (nonmammalian vertebrates being particularly relevant) have effective hemostatic mechanisms, none of which depends upon a megakaryocyte/platelet axis. We must ask, therefore, what is the biological advantage that led to the establishment and persistence of this cell lineage in mammals? Members of the vertebrate class Mammalia are characterized by body hair, mammary glands, and viviparous birth, except in the egg-laying monotremes. The presence of a placenta is also characteristic of most, but not all, pregnant female mammals. The two variations in birth mechanism, oviparous birth in monotremes and viviparous but nonplacental pregnancy in marsupials, present an opportunity to explore the potential association between placental pregnancy and platelet formation in mammals.

The Evolution of Mammalian Platelets

15

1

Fig. 1.25 Decoration of the extracellular blood clot by lipopolysaccharide (LPS). FITC-LPS (E. coli O55:B5) decorates the fibrin fibrils of the human clot prepared from platelet-depleted plasma (left) and the coagulin fibrils of the Limulus clot (right). The Limulus clot was also immunostained with a rabbit anticoagulin antibody and DyLight 549 Goat antirabbit whole IgG second antibody to show the location of the coagulin fibrils of the blood clot. (From Armstrong, et al.,127 with permission.)

Marine Mammals Marine mammals are unique because, with the exception of the duckbilled platypus, these are the only mammals that live in a marine environment. The platelets of the northern elephant seal (Mirounga angustirostris), collected in sodium citrate, demonstrate decreased responsiveness to thrombin, ADP, and ristocetin in comparison to platelets from humans and other mammals; shape change and aggregation were not produced by collagen or epinephrine in the absence of divalent cations.144 However, their platelets are morphologically similar to other mammalian platelets (Fig. 1.26). Similarly the

responsiveness of the platelets of the killer whale (Orcinus orca) to selected agonists was reduced, compared to human platelets, in experiments that utilized platelet-rich plasma prepared in sodium citrate.145 Only limited data are available for platelet counts in marine mammals.146–151 Platelet counts in selected marine mammals are shown in Table 1.3. Megakaryocytes are present in the bone marrow of the sea lion (Zalophus californianus) and are detectable with a polyclonal antihuman factor VIII-related antigen/von Willebrand factor antibody (Levin, J., unpublished observations).

Order Monotremata Monotremes, considered the most primitive form of mammals, have birdlike and reptilian features. The females lay eggs. Monotremes are represented by the aquatic duckbilled platypus and insectivorous echidna (spiny anteater). One report

TABLE 1.3 Platelet Counts in Selected Marine Mammals

Fig. 1.26 Representative field of unstimulated elephant seal platelets (Mirounga angustirostris). The cells are discoid, with tannic acid labeled membranous invaginations throughout the cells, suggestive of an OCS. Alpha granules (AG) are randomly dispersed. Microtubules (MT) are visible in cross-section. Glycogen granules (GLY) are dispersed throughout the cells, and mitochondria (M) are also seen (
21,000). (From Field et al.,144 with permission.)

Name

Ordera

Platelet Count (×109/L)b

Commerson’s dolphin Common dolphin Beluga whale Pilot whale White-sided dolphin Killer whale Bottlenose dolphin Northern fur seal Steller’s sea lion Northern elephant seal Harp seal Harbor seal Gray seal California sea lion Florida manatee Polar bear

Cetacea Cetacea Cetacea Cetacea Cetacea Cetacea Cetacea Pinnipedia Pinnipedia Pinnipedia Pinnipedia Pinnipedia Pinnipedia Pinnipedia Sirenia Carnivora

185 (10) 80 (2) 106 (16) 85(2) 120 (10) 163 (18) 165 (96) 428 (17) 243 (5) 437 (149) 500 (12) 314 (24) 378 (9) 280 (26) 283 (n.a.) 317 (n.a.)

n.a., not available. The order (subclass) within the class Mammalia is shown. b Mean platelet count. The number of animals studied is shown in parentheses. Data from Reidarson et al.147 (Almost all the published platelet counts of marine mammals have been provided by the clinical laboratory of Sea World.)

a

16

PART I Platelet Biology

on echidna platelets152 described two possible types: “elongated, spindle-shaped structures with a tendency to intertwine together or as normal platelets with spreading and aggregating activity” (p. 218). Hawkey152 suggested that the presence of two types of hemostatic cells in the echidna might indicate a link between the spindle-shaped thrombocytes of nonmammalian vertebrates and typical mammalian platelets. Platelet counts in echidna were 200 to 250
109/L. Another study153 of the echidna reported platelet levels of approximately 500 to 650
109/L, and that “giant multinucleated cells with a few platelet-like bodies within the cytoplasm” were present in the bone marrow (p. 1133). These giant cells were believed to be megakaryocytes. Other studies of the echidna Tachyglossus aculeatus reported platelet counts of 300 to 320
109/L154 and 205 to 682
109/L (18 animals).155 Platelet levels of approximately 400 to 450
109/L were reported for the duckbilled platypus Ornithorhynchus anatinus.156 The same investigators157 described platypus platelets as “anucleate, circular and 2 to 5 μm in diameter, with occasional large platelets (up to 8 μm) seen” (p. 423). Their electron microscopy studies demonstrated a homogeneous population of cells with typical platelet organelles and ultrastructure, including parallel bundles of microtubules.157 This detailed report emphasized that platypus platelets were similar in appearance and size to those of other mammals, including marsupials. The two types of platelets that Hawkey152 suggested were present in the echidna were not observed. Another investigation, based on 56 platypuses, reported that platelet levels ranged from 315 to 2144
109/L.155 Other studies established that the spleen was the primary hematopoietic organ in the platypus; there was a complete absence of detectable megakaryocytes in the bone marrow.158 Previous attempts by this author (J. L.) to obtain specimens of platypus bone marrow to study megakaryocytes have failed because of Australian and U.S. regulations restricting export and import of specimens from endangered wildlife species.

Order Marsupialia Marsupials give birth to live but very immature young, after a nonplacental pregnancy. This order is represented by the opossum, kangaroo, wombat, and bandicoot. An extensive histochemical study of the blood cells of the marsupial Trichosurus vulpecula, the Australian brush-tailed possum, provided multiple photographs of typical mammalian platelets with essentially the same histochemical characteristics as human platelets.159 Overall platelet size was approximately the same as that of human platelets, but a greater proportion of large platelets was reported to be present. No quantitative data were provided. Opossum platelets are shown in Fig. 1.27. For comparison, electron micrographs of platelets in other mammalian orders are shown in Fig. 1.28. In other investigations, platelet levels in five different species of marsupials ranged from approximately 200 to 500
109/L152,153,160,161 and in a sixth species (Setonix brachyurus, the quokka) from 425 to 1180
109/L (Table 1.4). The mean platelet count was reported to be 153
109/L in the common wombat (23 animals) and to range from 136 to 485
109/L in the red-necked wallaby.162 (Table 1.4). Release of serotonin, a characteristic of mammalian platelets, was also demonstrated for marsupial platelets.160 In two Australian marsupial species whose bone marrow has been studied, megakaryocytes were described, but not illustrated.153 Microscopic studies of the liver of the North American opossum Didelphis Virginia revealed the presence of significant numbers of megakaryocytes in “pouch-young” animals163 (These animals remain attached to the nipple for at least 60 days and only afterwards leave the pouch). Throughout this period, almost all megakaryocytes demonstrated one to four discrete nonlobulated nuclei, although the number of nuclei per cell increased with age. Lobulated nuclei were rare.163 The number of megakaryocytes in the liver decreased with age.163,164 As the animals increased in age, mature megakaryocytes appeared, and extensive demarcation membranes

Fig. 1.27 Transmission electron micrographs of opossum (Didelphis marsupialis virginiana) platelets. Nonplacental mammals have platelets, as do all mammals. Left: a circumferential band of microtubules (MT) is clearly shown, and an open canalicular system (CS) is present. A large granule (G) and scattered glycogen particles (GLY) are present. Right: a large mass of wavy fibrillar material (WFM) is present. This fibrillar material was observed in approximately 10% of all platelets; its composition is unknown. (From Lewis,35 with permission.)

The Evolution of Mammalian Platelets

17

1

Fig. 1.28 Transmission electron micrographs of platelets. Left: hedgehog (Erinaceus europaeus) platelets. These platelets contain many granules. A dense canalicular system (DCS), a Golgi apparatus (GOLGI), and mitochondria (M) are present. Bottom: two monkey (Macaca mulatta) platelets. These contain large clumps of glycogen (GLY) and an open canalicular system (OCS). Microtubules (MT), mitochondria (M), dense bodies (DB), and α-granules (a) are also present. Right: Indian elephant (Elephas maximus) platelets. α-Granules (a-gran), dense bodies (DB), glycogen particles (GLY), and microtubules (MT) are shown. An open canalicular system is also present. (From Lewis,35 with permission.)

TABLE 1.4 Platelet Counts in Selected Mammals Name

Ordera

Platelet Count (×109/L)b

Echidna Quokka Wallaby Opossum Hedgehog Bat Armadillo Porpoise Seal Elephant Manatee Baboon Chimpanzee Monkey

Monotremata Marsupialia Marsupialia Marsupialia Insectívora Chiroptera Edentata Cetacea Pinnipedia Proboscidea Sirenia Primate Primate Primate

549 (3) 1180 (1) 390 (1) 498 (9) 113 (2) 819 (5) 357 (6) 136 (12) 651 (5) 620 (6) 347 (5) 299 (7) 406 (5) 510 (9)

a

The order (subclass) within the class Mammalia is shown. Mean platelet count. The number of animals studied is shown in parentheses. Data were derived from Lewis.35 b

became clearly apparent. As is the pattern for mammalian megakaryocytes, maturation of the nucleus generally preceded cytoplasmic maturation.164 Concomitantly, megakaryocytopoiesis occurred in the hepatic sinusoids. Neither paper described the bone marrow or spleen as playing a role in the production of platelets. Importantly, low ploidy megakaryocytes were capable of producing platelets, as has been shown for the megakaryocytes in the spleens of mice, in which the bone marrow had been totally ablated.165 Fig. 1.29 presents micrographs of the bone marrow of the South American opossum (Monodelphis domestica), which provide multiple examples of megakaryocytes in this marsupial.

Platelet Levels Interestingly, in most of the marsupials and echidnas studied, the previously described inverse relationship between mammalian body size and platelet level in a limited group of animals was not evident.166 On the basis of the previously published nomogram and platelet levels in some additional mammals,

18

PART I Platelet Biology

Fig. 1.29 Specimens of bone marrow from the South American opossum (Monodelphis domestica) stained with Wright-Giemsa. The megakaryocytes are the large, multinucleated cells, with light blue or pinkish cytoplasm. (A) Two adjacent megakaryocytes (indicated by the arrows; original magnification
100). (B) The same two megakaryocytes are shown at a higher power (original magnification
250). (C) A single megakaryocyte is detectable (original magnification
250). (D) A very large megakaryocyte is shown (original magnification
250). The black bars in the right lower corner of each panel indicate 10 μm (shorter bar) and 50 μm (longer bar). Previous reports also described marsupial megakaryocytes as multinucleated cells.144, 154, 155 This is in important contrast to the megakaryocytes of other mammals, such as human and rodent, which contain a single, multilobed, polyploid nucleus.

significantly higher platelet counts would have been expected in the smaller monotremes and marsupials. Perhaps the failure of this relationship to hold reflects the primitive nature of these two mammalian orders. Platelet counts in selected mammals are shown in Table 1.4, and summarized elsewhere.167–169 Mean platelet volume is not related to body mass.168–170 During hibernation of 13-lined ground squirrels (Ictidomys tridecemlineatus), although there was a three-fold decrease in megakaryocytes and a 10-fold decrease in platelet levels, there was no differential expression of hematopoietic genes involved in megakaryocyte production.171 Nevertheless, the mean platelet volume of both hibernating and aroused squirrels was larger than in nonhibernating animals, suggesting that platelet production had continued during hibernation. In comparison, red blood cell levels remained unchanged during hibernation.

A Comparison of Nonplacental and Placental Mammals Blood coagulation has been described in both monotremes and marsupials as resembling human blood coagulation; clot retraction, which is consistent with normal platelet function, also has been observed in both orders.153,160 Based on the previous comparisons of the hemostatic systems of the aplacental and placental mammals, it appears that there is no specific association between either the development of a placenta during pregnancy (or the occurrence of live birth) or the appearance of a markedly different hemostatic mechanism in

mammals and the presence of megakaryocytes and platelets. Therefore, the suggestion that the evolution of platelets resulted from unique hemostatic requirements imposed by placental birth cannot be supported.172 The cause of the possible appearance of the megakaryocyte/platelet lineage 166 MYA (million years ago), approximately 100 MYA prior to the appearance of placental mammals, remains a mystery (Fig. 1.30).173 Nevertheless, a potential role for platelets during pregnancy in mammals has been described.174 The preimplantation embryo produces platelet-activating factor (PAF), which has been described as then activating platelets in the microvascular bed of the oviduct in mice.174 These same investigators suggested that platelets may contribute to the establishment of early pregnancy, because administration of inhibitors of PAF to mice reduced the number of implantation sites. Furthermore, they concluded that activated platelets may produce a range of molecules that support the attachment of the embryo to the uterine surface.

CONCLUSIONS There is, presumably, a biological advantage gained from the presence of polyploid megakaryocytes as the source for nonnucleated platelet progeny in mammals. However, this advantage has not yet been identified. It has been pointed out that the abolition of mitosis provides a basis for increasing RNA synthesis and therefore an increased potential for protein synthesis in the cell.175 In addition, cell growth and differentiation can

The Evolution of Mammalian Platelets

19

1

First appearance of mammalia

Monotremes

K-Pg extinction event

Marsupials

Hypothetical placental ancestor

Placentals

170 160 166

150

140

130

120

110

100

90

80

70 60 64.85 Time (millions of years ago)

50

40

30

20

10

0 Recent

Fig. 1.30 The timeline for the class Mammalia suggests that the megakaryocyte/platelet lineage may have first appeared 166 MYA (million years ago), approximately 100 MYA prior to the hypothetical placental ancestor. (From O’Leary,173 with permission.)

continue, uninterrupted by nuclear and cell divisions. Nagl175 has also suggested that regulation of specific gene activity in a polyploid cell is more efficient than in a group of diploid cells. An obvious benefit is the ability of a single megakaryocyte to produce many hundreds or thousands of platelets. However, augmented production can be achieved by other means: The bone marrow can markedly and adequately increase the production of red and white blood cells without resorting to a mechanism based on polyploid progenitors and cytoplasmic fragmentation. Furthermore, the many animal species with nucleated thrombocytes have seemingly adequate hemostasis, and as yet, platelets have not been found to be mandatory for any special feature of mammalian blood coagulation that is not present in nonmammals. However, the mechanism by which platelets are produced by megakaryocytes does allow for the rapid release of larger than normal platelets.176,177 These cells are more biologically active than platelets produced under steady-state conditions, and therefore may constitute an attempt to provide a maximally effective response to a pathophysiological emergency. Perhaps similarly, the plant thale cress, Arabidopsis thaliana genotype Columbia-4 employs endoreduplication following damage and overcompensates by increasing seed yield.178 The plant’s ability to increase its ploidy during regrowth helped mitigate the damage. The ability of hepatocytes to become polyploid is believed to play a role their ability to regenerate. Another unexplained element is the presence of high concentrations of acetyl cholinesterase (AChE) in the megakaryocytes of only some mammalian species, such as the mouse, rat, and cat, but not humans.179 Why should only select species have megakaryocytes that produce high concentrations of AChE? Mechanisms for the regulation of megakaryocytopoiesis and of platelet function appear identical regardless of the presence or absence of AChE in megakaryocytes. In addition, inhibitors of AChE do not have any detectable effect on platelet aggregation, platelet factor 3 availability, or plasma clot retraction.180 Elucidation of the evolutionary event or events that resulted in the appearance of mammalian megakaryocytes and platelets, as well as the potential biological advantage of this system, remains elusive. Collectively, c-Mpl,73,181 CD41,73,141,143, 181

other platelet surface markers,44, 138,139,141 responses to platelet agonists,54,63,72 and polyploid hematopoietic precursors72 have been described in nonmammalian vertebrates. Some thrombocytes contain serotonin, as do platelets.52,53 Responsiveness to thrombopoietin (TPO) has been demonstrated73,181 and it has been concluded that TPO as a regulator of thrombopoiesis is evolutionally conserved in vertebrates.181 Therefore, genetic components necessary for generation of the megakaryocyte/platelet lineage have been present long before the appearance of mammals. What series of events finally resulted in the appearance of megakaryocytes in monotremes, the most primitive order of mammals? Because both monotremes, which are egg-laying mammals, and marsupials, which have a nonplacental pregnancy, possess megakaryocytes and platelets, it is apparent that neither live birth nor the presence of a placenta accounts for the evolution of platelets in mammals. It seems unlikely that the diaphragm, present only in mammals, could have triggered this striking evolutionary step. Comparative molecular genetic studies are likely to provide further insights.

Acknowledgments I express my personal and professional gratitude to the late Dr. Frederik B. Bang for introducing me to Limulus over 50 years ago, and for providing the intellectual guidance that made it possible for me to begin the studies that constitute a significant component of this chapter. I also wish to acknowledge the superb and supportive environment of the Marine Biological Laboratory, Woods Hole, Massachusetts, which nurtured my interests in comparative hemostasis, and the many staff members and scientists at M.B.L. who provided me with the information and techniques that I required to pursue my experimental goals. I thank the staff of the Medical Library at the San Francisco Veterans Administration Medical Center for their consistent and effective support of my efforts to access a widely scattered literature, much of which was published decades before the advent of databases. The ongoing unfailing ability of Ms. Nadine Walas to obtain copies of papers in obscure journals or in volumes published at the beginning of the 20th century is especially appreciated. The outstanding skills of Mr. Edgardo Caballero, the medical photographer in the Medical Media section at the San Francisco VAMC, made it possible to prepare many of the figures, especially the color plates.

20

PART I Platelet Biology

This chapter is dedicated to the late Dr. Jessica H. Lewis, whose instructive studies provided many of the figures that appear in this analysis.

REFERENCES 1. Levin J, Bang FB. A description of cellular coagulation in the Limulus. Bull Johns Hopkins Hosp 1964;115:337–45. 2. Kenny DM, Belamarich FA, Shepro D. Aggregation of horseshoe crab (Limulus polyphemus) amebocytes and reversible inhibition of aggregation by EDTA. Biol Bull 1972;143:548–67. 3. Takahashi H, Azumi K, Yokosawa H. Hemocyte aggregation in the solitary ascidian Halocynthia roretzi: plasma factors, magnesium ion, and met-lys-bradykinin induce the aggregation. Biol Bull 1994;186:247–53. 4. Goffinet G, Gr
egoire C. Coagulocyte alterations in clotting hemolymph of Carausius morosus L. Arch Int Physiol Biochim 1975;83:707–22. 5. Levin J. The horseshoe crab: a model for Gram-negative sepsis in marine organisms and humans. In: Levin J, Buller HR, ten Cate JW, van Deventer SJH, Sturk A, editors. Bacterial endotoxins. Pathophysiological effects, clinical significance, and pharmacological control. New York: Alan R. Liss; 1988. p. 3–15. 6. Copeland DE, Levin J. The fine structure of the amebocyte in the blood of Limulus polyphemus. I. Morphology of the normal cell. Biol Bull 1985;169:449–57. 7. M€ urer EH, Levin J, Holme R. Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab. J Cell Physiol 1975;86:533–42. 8. Chen JH, Bayne CJ. Bivalve mollusc hemocyte behaviors: characterization of hemocyte aggregation and adhesion and their inhibition in the California mussel (Mytilus californianus). Biol Bull 1995;188:255–66. 9. Gr
egoire C. Haemolymph coagulation in insects and taxonomy. Bull K Belg Inst Nat Wet 1984;55:3–48. 10. Zachary D, Hoffmann JA. The haemocytes of Calliphora erythrocephala (Meig.) (Diptera). Z Zellforsch Mikroskop Anat (Vienna) 1973;141:55–73. 11. Zachary D, Br
ehelin M, Hoffmann JA. Role of the “Thrombocytoids” in capsule formation in the Dipteran Caliphora erythrocephala. Cell Tissue Res 1975;162:343–8. 12. Ratnoff OD. The evolution of hemostatic mechanisms. Perspect Biol Med 1987;31:4–33. 13. Needham AE. Haemostatic mechanisms in the invertebrata. Symp Zool Soc Lond 1970;27:19–44. 14. Dales RP, Dixon LRJ. Polychaetes. In: Ratcliffe NA, Rowley AF, editors. Invertebrate blood cells. vol. 1. London: Academic Press; 1981. p. 35–74. 15. Ravindranath MH. Haemocytes in haemolymph coagulation of arthropods. Biol Rev 1980;55:139–70. 16. Levin J, Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull Johns Hopkins Hosp 1964;115:265–74. 17. Levin J, Bang FB. Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin. Thromb Diath Haemorrh 1968;19:186–97. 18. Madaras F, Parkin JD, Castaldi PA. Coagulation in the sand crab (Ovalipes bipustulatus). Thromb Haemost 1979;42:734–42. 19. Hose JE, Martin GG, Gerard AS. A decapod hemocyte classification scheme integrating morphology, cytochemistry, and function. Biol Bull 1990;178:33–45. 20. Spurling NW. Comparative physiology of blood clotting. Comp Biochem Physiol 1981;68A:541–8. 21. Levin J. The role of amebocytes in the blood coagulation mechanism of the horseshoe crab Limulus polyphemus. In: Cohen W, editor. Blood cells of marine invertebrates: experimental systems in cell biology and comparative physiology. New York: Alan R. Liss; 1985. p. 145–63. 22. Young NS, Levin J, Prendergast RA. An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation. J Clin Invest 1972;51:1790–7. 23. Cerenius L, S€ oderh€all K. Coagulation in invertebrates. J Innate Immun 2011;3:3–8. 24. Schneider W, Gattermann N. Megakaryocytes: origin of bleeding and thrombotic disorders. Eur J Clin Invest 1994;24:16–20. Suppl. 1.

25. Ellis AE. Leukocytes and related cells in the plaice. Pleuronectes platessa. J Fish Biol 1976;8:143–56. 26. Ellis AE. The leukocytes of fish: a review. J Fish Biol 1977;11:453–91. 27. Westermann JEM. Light microscopic study and identification of thrombocytes of peripheral blood of the turtle. Rev Can Biol 1974;33:255–67. 28. Gardner GR, Yevich PP. Studies on the blood morphology of three estuarine cyprinodontiform fishes. J Fish Res Board Can 1969;26:433–47. 29. Saunders DC. Differential blood cell counts of 121 species of marine fishes in Puerto Rico. Trans Am Microsc Soc 1966;85:427–49. 30. Tavares-Dias M, Moraes FR. Morphological, cytochemical, and ultrastructural study of thrombocytes and leukocytes in neotropical fish, Brycon orbignyanus Valenciennes, 1850 (Characidae Bryconinae). J Submicrosc Cytol Pathol 2006;38:209–15. 31. Shigdar S, Harford A, Ward AC. Cytochemical characterisation of the leucocytes and thrombocytes from Murray cod (Maccullochella peelii peelii, Mitchell). Fish Shellfish Immunol 2009;26:731–6. 32. Zinkl JG, Cox WT, Kono CS. Morphology and cytochemistry of leucocytes and thrombocytes of six species of fish. Comp Haematol Int 1991;1:187–95. 33. Burrows AS, Fletcher TC, Manning MJ. Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes. J Appl Ichthyol 2001;17:77–84. 34. Hine PM, Wain JM, Lester RJ,G. Observations on the blood of the Australian lungfish, Neoceratodus forsteri Klefft. II. Enzyme cytochemistry of blood cells, peritoneal macrophages and melanomacrophages. Aust J Zool 1990;38:145–54. 35. Lewis JH. Comparative hemostasis in vertebrates. New York: Plenum Press; 1996. p. 3–322. 36. Grant M, Beeler DL, Spokes KC, Chen J, Dharaneeswaran H, Sciuto TE, Dvorak AM, Interlandi G, Lopez JA, Aird WC. Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis. Blood 2017;130:2548–58. 37. Esteban MA, Muñoz J, Meseguer J. Blood cells of sea bass (Dicentrarchus labrax L.). Flow cytometric and microscopic studies. Anat Rec 2000;258:80–9. 38. Ferguson H,W. The ultrastructure of plaice (Pleuronectes platessa) leucocytes. J Fish Biol 1976;8:139–42. 39. Rowley AF, Hill DJ, Ray CE, Munro R. Haemostasis in fish—an evolutionary perspective. Thromb Haemost 1997;77:227–33. 40. Tavares-Dias M, Ono EA, Pilarski F, Moraes FR. Can thrombocytes participate in the removal of cellular debris in the blood circulation of teleost fish? A cytochemical study and ultrastructural analysis. J Appl Ichthyol 2007;23:709–12. 41. Belamarich FA, Fusari MH, Shepro D, Kien M. In vitro studies of aggregation of non-mammalian thrombocytes. Nature 1966;212:1579–80. 42. Soslau G, Prest PJ, Class R, George R, Paladino F, Violetta G. Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood. Comp Biochem Physiol Part B 2005;142:353–60. 43. Thattaliyath B, Cykowski M, Jagadeeswaran P. Young thrombocytes initiate the formation of arterial thrombi in zebrafish. Blood 2005;106:118–24. 44. Schmaier A, Stalker TJ, Runge JJ, Lee D, Nagaswami C, Mericko P, Chen M, Clich
e S, Gari
epy C, Brass LF, Hammer DA, Weisel JW, Rosenthal K, Kahn ML. Occlusive thrombi arise in mammals but not birds in response to arterial injury: evolutionary insight into human cardiovascular disease. Blood 2011;118: 3661–9. 45. Campbell TW. Normal hematology of psittacines. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 1155–63. 46. Stalsberg H, Prydz H. Studies on chick embryo thrombocytes. I. Morphology and development. Thromb Diath Haemorrh 1963;9:279–90. 47. Maxwell MH. An ultrastructural comparison of the mononuclear leucocytes and thrombocytes in six species of domestic bird. J Anat 1974;117:69–80. 48. Bohls RL, Smith R, Ferro PJ, Silvy NJ, Li Z, Collisson EW. The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes. Dev Comp Immunol 2006;30:843–50.

The Evolution of Mammalian Platelets 49. Bertram EM. Characterisation of duck thrombocytes. Res Vet Sci 1998;64:267–70. 50. Lucas AM, Jamroz C. Atlas of avian hematology (agriculture monograph 25). Washington, DC: US Department of Agriculture; 1961. 51. Carlson HC, Sweeny PR, Tokaryk JM. Demonstration of phagocytic and trephocytic activities of chicken thrombocytes by microscopy and vital staining techniques. Avian Dis 1968;12:700–15. 52. Kuruma I, Okada T, Kataoka K, Sorimachi M. Ultrastructural observation of 5-hydroxytryptamine-storing granules in the domestic fowl thrombocytes. Z Zellforsch Mikrosk Anat 1970;108:268–81. 53. Maurer-Spurej E. Circulating serotonin in vertebrates. Cell Mol Life Sci 2005;62:1881–9. 54. Wachowicz B, Krajewski T. The released proteins from avian thrombocytes. Thromb Haemost 1979;42:1289–95. 55. Daimon T, Mizuhira V, Takahashi I, Uchida K. The surface connected canalicular system of carp (Cyprinus carpio) thrombocytes: its fine structure and three-dimensional architecture. Cell Tissue Res 1979;203:355–65. 56. Daimon T, Mizuhira V, Uchida K. Fine structural distribution of the surface-connected canalicular system in frog thrombocytes. Cell Tissue Res 1979;201:431–9. 57. Daimon T, Uchida K. Electron microscopic and cytochemical observations on the membrane systems of the chicken thrombocyte. J Anat 1978;125:11–21. 58. G€ ohler S, Greven H. Ultrastructure of mature thrombocytes in the peripheral blood of Salamandra salamandra (L.) (Amphibia, Urodela). Z Mikrosk Anat Forsch (Leipzig) 1985;99:369–77. 59. Veiga ML, Egami MI, Ranzani-Paiva MJT, Rodrigues EL. Morphological and ultrastructural study of the thrombocytes and leukocyte granulocytes of Salminus maxillosus (Characiformes, Characidae). J Submicrosc Cytol Pathol 2002;34:397–402. 60. Stenberg PE, Levin J. Mechanisms of platelet production. Blood Cells 1989;15:23–47. 61. Zucker-Franklin D, Benson KA, Myers KM. Absence of a surfaceconnected canalicular system in bovine platelets. Blood 1985;65:241–4. 62. du Plessis L, Stevens K. Blood platelets of the African elephant. J Comp Pathol 2002;127:208–10. 63. Lee K-G, Miler T, Anastassov I, Cohen WD. Shape transformation and cytoskeletal reorganization in activated non-mammalian thrombocytes. Cell Biol Int 2004;28:299–310. 64. Pica A, Della Corte F, Grimaldi MC, D’Ippolito S. The blood cells of the common lizard (Podarcis S. sicula Raf.). Arch Ital Anat Embriol 1986;91:301–20. 65. Pica A, Lodato A, Grimaldi MC, Della Corte F. Morphology, origin and functions of the thrombocytes of Elasmobranchs. Arch Ital Anat Embriol 1990;95:187–207. 66. Pica A, Lodato A, Grimaldi MC, Della Corte F, Galderisi U. A study of the bone marrow precursors and hemoglobin of the blood cells of the red-legged partridge (Alectoris rufa rufa L.). Ital J Anat Embryol 1993;98:277–92. 67. D’Ippolito S, Pica A, Grimaldi MC, Della Corte F. The blood cells and their precursors in the hemopoietic organs of the dogfish Scyliorhynus stellaris L. Arch Ital Anat Embriol 1985;90:31–46. 68. Jain NC. Blood cells and parasites of birds. In: Jain NC, editor. Schalm’s veterinary hematology. 4th ed. Philadelphia: Lea & Febiger; 1986. plate XXV. 69. Martínez-Silvestre A, Marco I, Rodriguez-Dominguez MA, Lavín S, Cuenca R. Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi). Res Vet Sci 2005;78:127–34. 70. Canfield PJ. Characterization of the blood cells of Australian crocodiles (Crocodylus porosus [Schneider] and C. johnstoni [Krefft]). Zbl Anat Histol Embryol 1985;14:269–88. 71. Daimon T, Gotoh Y, Uchida K. Electron microscopic and cytochemical studies of the thrombocytes of the tortoise (Geoclemys reevesii). J Anat 1987;153:185–90. 72. Tanizaki Y, Ishida-Iwata T, Obuchi-Shimoj M, Kato T. Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies. Exp Hematol 2015;43:125–36. 73. Tanizaki Y, Ichisugi M, Obuchi-Shimoji M, Ishida-Iwata T, Tahara-Mogi A, Meguro-Ishikawa M, Kato T. Thrombopoietin induces production of nucleated thrombocytes from liver cells

74. 75.

76. 77. 78. 79. 80. 81.

82. 83. 84. 85. 86. 87. 88. 89.

90. 91.

92. 93.

94. 95. 96. 97. 98.

21

in Xenopus laevis. Sci Rep 2015;5:18519. https://doi.org/ 10.1038/srep18519. Hawkey CM. General summary and conclusions. Symp Zool Soc Lond 1970;27:217–29. Belamarich FA. Hemostasis in animals other than mammals: the role of cells. In: Spaet TH, editor. Progress in Hemostasis and Thrombosis. vol. 3. New York: Grune and Stratton; 1976. p. 191–209. Hewson W. Gulliver FRSG, editor. The works of William Hewson. London: C. and J. Adlard; 1846. p. 211–44. Iwanaga S, Kawabata SI, Muta T. New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. J Biochem 1998;123:1–15. Armstrong PB, Rickles FR. Endotoxin-induced degranulation of the Limulus amebocyte. Exp Cell Res 1982;140:15–24. Dumont JN, Anderson E, Winmer G. Some cytologic characteristics of the hemocytes of Limulus during clotting. J Morphol 1966;119:181–208. Ornberg RL, Reese TS. Beginning of exocytosis captured by rapidfreezing of Limulus amebocytes. J Cell Biol 1981;909:40–54. Tablin F, Levin J. The fine structure of the amebocyte in the blood of Limulus polyphemus. II. The amebocyte cytoskeleton: a morphological analysis of native, activated, and endotoxin-stimulated amebocytes. Biol Bull 1988;175:417–29. S€ oderh€all K, Levin J, Armstrong PB. The effects of β1,3-glucans on blood coagulation and amebocyte release in the horseshoe crab, Limulus polyphemus. Biol Bull 1985;169:661–74. Inamori K, Ariki S, Kawabata S. A Toll-like receptor in horseshoe crabs. Immunol Rev 2004;198:106–15. Rickles FR, Rick P, Armstrong PB, Levin J. Binding studies of radioactive lipopolysaccharide with Limulus amebocytes. Prog Clin Biol Res 1979;29:203–7. Coates CJ, Whalley T, Nairn J. Phagocytic activity of Limulus polyphemus amebocytes in vitro. J Invertebr Pathol 2012;111: 205–10. Zander DMW, Klinger MHF. The blood platelets contribution to innate host defense—What they have learned from their big brothers. Biotechnol J 2009;4:914–26. Rudkin DM, Young GA, Nowlan GS. The oldest horseshoe crab: a new Xiphosurid from Late Ordovician Konservat-Lagerst€atten deposits, Manitoba, Canada. Palaeontology 2008;51(Pt 1):1–9. Hill DJ, Rowley AF. Are integrins involved in the aggregatory and phagocytic behaviour of fish haemostatic cells? J Exp Biol 1998;201:599–608. Levin J. The role of amebocytes in the blood coagulation mechanism of the horseshoe crab Limulus polyphemus. In: Cohen W, editor. Blood cells of marine invertebrates: experimental systems in cell biology and comparative physiology. New York: Alan R. Liss; 1985. p. 145–63. Glynn MF, Movat HZ, Murphy EA, Mustard JF. Study of platelet adhesiveness and aggregation, with latex particles. J Lab Clin Med 1965;65:179–201. Levin J. Blood coagulation in the horseshoe crab (Limulus polyphemus): a model for mammalian coagulation and hemostasis. In: Animal models of thrombosis and hemorrhagic diseases. Washington, DC: US Department of Health, Education and Welfare; 1976. p. 87–96. Publication No. (NIH) 76-982. Lewis JC, Maldonado JE, Mann KG. Phagocytosis in human platelets: localization of acid phosphatase-positive phagosomes following latex uptake. Blood 1976;47:833–40. Youssefian T, Drouin A, Mass
e JM, Guichard J, Cramer EM. Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation. Blood 2002;99:4021–9. White JG. Platelets are covercytes, not phagocytes: uptake of bacteria involves channels of the open canalicular system. Platelets 2005;16:121–31. Meseguer J, Esteban MA, Rodríguez A. Are thrombocytes and platelets true phagocytes? Microsc Res Tech 2002;57:491–7. Springer GF, Adye JC. Endotoxin-binding substances from human leukocytes and platelets. Infect Immun 1975;12:978–86. Clawson CC. Platelet interaction with bacteria. III. Ultrastructure. Am J Pathol 1973;70:449–72. MacIntyre DE, Allen AP, Thorne KJI, Glauert AM, Gordon JL. Endotoxin-induced platelet aggregation and secretion. I.

1

22

99. 100. 101. 102. 103. 104.

105. 106. 107.

108.

109. 110.

111. 112.

113.

114. 115. 116. 117. 118. 119. 120.

121.

PART I Platelet Biology Morphological changes and pharmacological effects. J Cell Sci 1977;28:211–23. Levin J. Bleeding with infectious diseases. In: Ratnoff OD, Forbes CD, editors. Disorders of hemostasis. 3rd ed. Philadelphia: WB Saunders; 1996. p. 339–56. Clawson CC. Platelets in bacterial infections. In: Joseph M, editor. Immunopharmacology of platelets. London: Harcourt Brace; 1995. p. 83–124. Yeaman MR. The role of platelets in antimicrobial host defense. Clin Infect Dis 1997;25:951–70. Andonegui G, Kerfoot SM, McNagny K, Ebbert KVJ, Patel KD, Kubes P. Platelets express functional toll-like receptor-4. Blood 2005;106:2417–23. Cognasse F, Hamzeh H, Chavarin P, Acquart S, Genin C, Garraud O. Evidence of Toll-like receptor molecules on human platelets. Immunol Cell Biol 2005;83:196–8. Scott T, Owens MD. Thrombocytes respond to lipopolysaccharide through Toll-like receptor-4, and MAP kinase and NF-κB pathways leading to expression of interleukin-6 and cyclooxygenase2 with production of prostaglandin E2. Mol Immunol 2008;45:1001–8. Klinger MHF, Jelkmann W. Role of blood platelets in infection and inflammation. J Interf Cytokine Res 2002;22:913–22. Clawson CC, Rao GHR, White JG. Platelet interaction with bacteria. IV. Stimulation of the release reaction. Am J Pathol 1975;81:411–9. Yeaman MR, Bayer AS, Koo SP, Foss W, Sullam PM. Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action. J Clin Invest 1998;101:178–87. Hawiger J, Steckley S, Hammond D, Cheng C, Timmons S, Glick AD, Des Prez RM. Staphylococci-induced human platelet injury mediated by protein A and immunoglobulin G Fc fragment receptor. J Clin Invest 1979;64:931–7. Siboo IR, Cheung AL, Bayer AS, Sullam PM. Clumping factor A mediates binding of Staphylococcus aureus to human platelets. Infect Immun 2001;69:3120–7. Zimmerman TS, Spiegelberg HL. Pneumococcus-induced serotonin release from human platelets. Identification of the participating plasma/serum factor as immunoglobulin. J Clin Invest 1975;56:828–34. Bik T, Sarov I, Livne A. Interaction between vaccinia virus and human blood platelets. Blood 1982;59:482–7. Deng L, Bensing BA, Thamadilok S, Yu H, Lau K, Chen X, Stefan Ruhl S, Sullam PM, Ajit Varki A. Oral Streptococci utilize a Sigleclike domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood. PLoS Pathog 2014;10(12). e1004540. https://doi.org/10.1371/journal.ppat.1004540. Seo HS, Xiong YQ, Mitchell J, Seepersaud R, Bayer AS, Sullam PM. Bacterophage lysin mediates the binding of Streptococcus mitis to human platelets through interaction with fibrinogen. PLoS Pathog 2010;6:. e1001047. Sullam PM, Jarvis GA, Valone FH. Role of immunoglobulin G in platelet aggregation by viridans group streptococci. Infect Immun 1988;56:2907–11. Herzberg MC, Brintzenhofe KL, Clawson CC. Aggregation of human platelets and adhesion of Streptococcus sanguis. Infect Immun 1983;39:1457–69. Kurpiewski GE, Forrester LJ, Campbell BJ, Barrett JT. Platelet aggregation by Streptococcus pyogenes. Infect Immun 1983;39:704–8. Capron A, Ameisen JC, Joseph M, Auriault C, Tonnel AB, Caen J. New functions for platelets and their pathological implications. Int Arch Allergy Appl Immunol 1985;77:107–14. Pancr
e V, Auriault C. Platelets in parasitic diseases. In: Joseph M, editor. Immunopharmacology of platelets. London: Harcourt Brace; 1995. p. 125–35. Ngaiza JR, Doenhoff MJ. Blood platelets and schistosome egg excretion. Proc Soc Exp Biol Med 1990;193:73–9. Pancr
e V, Mont
e D, Delanoye A, Capron A, Auriault C. Interleukin-6 is the main mediator of the interaction between monocytes and platelets in the killing of Schistosoma mansoni. Eur Cytokine Netw 1990;1:15–9. Lowenhaupt RW, Miller MA, Glueck HI. Platelet migration and chemotaxis demonstrated in vitro. Thromb Res 1973;3:477–87.

122. Lowenhaupt RW, Glueck HI, Miller MA, Kline DL. Factors which influence blood platelet migration. J Lab Clin Med 1977;90:37–45. 123. Czapiga M, Gao J-L, Kirk A, Lekstrom-Himes J. Human platelets exhibit chemotaxis using functional N-formyl peptide receptors. Exp Hematol 2005;33:73–84. 124. Armstrong PB. Motility of the Limulus blood cell. J Cell Sci 1979;37:169–80. 125. Gaertner F, Ahmad Z, Rosenberger G, Fan S, Nicolai L, Busch B, Yavuz G, Luckner M, Ishikawa-Ankerhold H, Hennel R, Benechet A, Lorenz M, Chandraratne S, Schubert I, Helmer S, Striednig B, Stark K, Janko M, B€ ottcher RT, Verschoor A, Leon C, Gachet C, Gudermann T, Mederos y Schnitzler M, Pincus Z, Iannacone M, Haas R, Wanner G, Kirsten Lauber K, Sixt M, Massberg S. Migrating platelets are mechano-scavengers that collect and bundle bacteria. Cell 2017;171:1368–82. 126. Chaikeeratisak V, Tassanakajon A, Armstrong PB. Interaction of pathogenic Vibrio bacteria with the blood clot of the Pacific White Shrimp, Litopenaeus vannamei. Biol Bull 2014;226:102–10. 127. Armstrong MT, Rickles FR, Armstrong PB. Capture of Lipopolysaccharide (Endotoxin) by the blood clot: a comparative study. PLoS ONE 2013;8(11). e80192. https://doi.org/10.1371/journal. pone.0080192. 128. Bayer AS, Sullam PM, Ramos M, Li C, Cheung AL, Yeaman MR. Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains. Infect Immun 1995;63:3634–41. 129. Quick AJ. Hemostasis as an evolutionary development. Thromb Diath Haemorrh 1967;18:1–11. 130. Laki K. Our ancient heritage in blood clotting and some of its consequences. Ann N Y Acad Sci 1972;202:297–307. 131. Page CP. Platelets as inflammatory cells. Immunopharmacology 1989;17:51–9. 132. Weyrich AS, Lindemann S, Zimmerman GA. The evolving role of platelets in inflammation. J Thromb Haemost 2003;1:1897–905. 133. Doolittle RF. Step-by-step evolution of vertebrate blood coagulation. Cold Spring Harb Symp Quant Biol 2009;74:35–40. 134. Gould SJ. Bushes and ladders in human evolution. In: Ever since Darwin. Reflections in natural history. New York: WW Norton; 1977. p. 56–62. 135. Gould SJ. The episodic nature of evolutionary change. In: The Panda’s thumb. More Reflections in natural history. New York: WW Norto; 1980. p. 179–85. 136. Ratcliffe NA, Millar DA. Comparative aspects and possible phylogenetic affinities of vertebrate and invertebrate blood cells. In: Rowley AF, Ratcliffe NA, editors. Vertebrate blood cells. Cambridge, UK: Cambridge University Press; 1988. p. 1–17. 137. Kunicki TJ, Newman PJ. Synthesis of analogs of human platelet membrane glycoprotein IIb-IIIa complex by chicken peripheral blood thrombocytes. Proc Natl Acad Sci U S A 1985;82:7319–23. 138. Lacoste-Eleaume AS, Bleux C, Qu
er
e P, Coudert F, Corbel C, Kanellopoulos-Langevin C. Biochemical and functional characterization of an avian homolog of the integrin GPIIb-IIIa present on chicken thrombocytes. Exp Cell Res 1994;213:198–209. 139. Ody C, Vaigot P, Qu
er
e P, Imhof BA, Corbel C. Glycoprotein IIbIIIa is expressed on avian multilineage hematopoietic progenitor cells. Blood 1999;93:2898–906. 140. O’Toole ET, Hantgan RR, Lewis JC. Localization of fibrinogen during aggregation of avian thrombocytes. Exp Mol Pathol 1994;61:175–90. 141. Passer BJ, Chen CH, Miller NW, Cooper MD. Catfish thrombocytes express an integrin-like CD41/CD61 complex. Exp Cell Res 1997;234:347–53. 142. Jagadeeswaran P, Sheehan JP, Craig FE, Troyer D. Identification and characterization of zebrafish thrombocytes. Br J Haematol 1999;107:731–8. 143. Lin HF, Traver D, Zhu H, Dooley K, Paw BH, Zon LI, Handin RI. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 2005;106:3803–10. 144. Field CL, Walker NJ, Tablin F. Northern elephant seal platelets: analysis of shape change and response to platelet agonists. Thromb Res 2001;101:267–77. 145. Patterson WR, Dalton LM, McGlasson DL, Cissik JH. Aggregation of killer whale platelets. Thromb Res 1993;70:225–31.

The Evolution of Mammalian Platelets 146. Bossart GD, Reidarson TH, Dierauf LA, Duffield DA. Clinical pathology. In: Dierauf LA, Gulland FMD, editors. CRC handbook of marine mammal medicine. 2nd ed. Boca Raton, FL: CRC Press; 2001. p. 383–436. 147. Reidarson TH. Cetacea (whales, dolphins, porpoises). In: Fowler ME, Miller RE, editors. Zoo and wild animal medicine. 5th ed. St. Louis: Saunders; 2003. p. 442–58. 148. Gage LJ. Pinnipedia (seals, sea lions, walruses). In: Fowler ME, Miller RE, editors. Zoo and wild animal medicine. 5th ed. St. Louis: Saunders; 2003. p. 459–75. 149. Murphy D. Sirenia. In: Fowler ME, Miller RE, editors. Zoo and wild animal medicine. 5th ed. St. Louis: Saunders; 2003. p. 476–81. 150. Walsh MT, Bossart GD. Manatee medicine. In: Fowler ME, Miller RE, editors. Zoo and wild animal medicine. 4th ed. St. Louis: Saunders; 1999. p. 507–516.0. 151. Reidarson TH, Duffield D, McBain J. Normal hematology of marine mammals. In: Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s veterinary hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 1164–73. 152. Hawkey CM. Comparative mammalian haematology: cellular components and blood coagulation of captive wild animals. London: William Heinemann Medical Books; 1975. p. 218–27. 153. Lewis JH, Phillips LL, Hann C. Coagulation and hematological studies in primitive Australian mammals. Comp Biochem Physiol 1968;25:1129–35. 154. Bolliger A, Backhouse TC. Blood studies on the echidna Tachyglossus aculeatus. Proc Zool Soc London 1960;135:91–7. Pt 1. 155. Booth RJ. Monotremata (echidna, platypus). In: Fowler ME, Miller RE, editors. Zoo and wild animal medicine. 5th ed. St. Louis: Saunders; 2003. p. 278–87. 156. Whittington RJ, Grant TR. Haematology and blood chemistry of the free-living platypus, Ornithorhynchus anatinus (Shaw) (Monotremata: Ornithorhynchidae). Aust J Zool 1983;31:475–82. 157. Canfield PJ, Whittington RJ. Morphological observations on the erythrocytes, leukocytes and platelets of free-living platypuses, Ornithorhynchus anatinus (Shaw) (Monotremata: Ornithorhynchidae). Aust J Zool 1983;31:421–32. 158. Tanaka Y, Eishi Y, Morris B. Splenic hemopoiesis of the platypus (Ornithorhynchus anatinus): evidence of primary hemopoiesis in the spleen of a primitive mammal. Am J Anat 1988;181:401–5. 159. Barbour RA. The leukocytes and platelets of a marsupial, Trichosurus vulpecula. A comparative morphological, metrical, and cytochemical study. Arch Histol Jpn 1972;34:311–60. 160. Fantl P, Ward HA. Comparison of blood clotting in marsupials and man. Aust J Exp Biol 1957;35:209–24. 161. Lewis JH. Comparative hematology: studies on opossums Didelphis marsupialis (Virginianus). Comp Biochem Physiol 1975;51A:275–80. 162. Holz P. Marsupialia (marsupials). In: Fowler ME, Miller RE, editors. Zoo and wild animal medicine. 5th ed. St. Louis: Saunders; 2003. p. 288–303. 163. Cutts JH, Leeson CR, Krause WJ. The postnatal development of the liver in a marsupial, Didelphis virginiana. J Anat 1973;115:327–46.

23

164. Paone DB, Cutts JH, Krause WJ. Megakaryocytopoiesis in the liver of the developing opossum (Didelphis virginiana). J Anat 1975;120:239–52. 165. Davis E, Corash L, Baker G, Mok Y, Hill RJ, Levin J. Splenic thrombopoiesis after bone marrow ablation with radiostrontium: a murine model. J Lab Clin Med 1990;116:879–88. 166. Nakeff A, Ingram M. Platelet count: volume relationships in four mammalian species. J Appl Physiol 1970;28:530–3. 167. Jain NC. Essentials of veterinary hematology. Philadephia: Lea & Febiger; 1993. p. 54–71. 168. Boudreaux MK, Ebbe S. Comparison of platelet number, mean platelet volume and platelet mass in five mammalian species. Comp Haematol Int 1998;8:16–20. 169. Ebbe S, Boudreaux MK. Relationship of megakaryocyte ploidy with platelet number and size in cats, dog, rabbits and mice. Comp Haematol Int 1998;8:21–5. 170. Jain NC. Essentials of veterinary hematology. Philadephia: Lea & Febiger; 1993. p. 105–32. 171. Cooper ST, Sell SS, Fahrenkrog M, Wilkinson K, Howard DR, Bergen H, Cruz E, Cash SE, Andrews MT, Hampton M. Effects of hibernation on bone marrow transcriptome in thirteen-lined ground squirrels. Physiol Genomics 2016;48:513–25. 172. Martin JF. Punctuated equilibrium in evolution and the platelet. Eur J Clin Investig 1994;24:291 (letter). 173. O’Leary MA. On the trail of the first placental mammals. Am Sci 2014;102:190–7. 174. Burton G, O’Neill C, Saunders DM. Platelets and pregnancy. In: Page CP, editor. The platelet in health and disease. London: Blackwell Scientific Publications; 1991. p. 191–209. 175. Nagl W. Functional significance of endo-cycles. In: Endopolyploidy and polyteny in differentiation and evolution. Amsterdam: NorthHolland Publishing; 1978. p. 154–7. 176. Stenberg PE, Levin J. Ultrastructural analysis of acute immune thrombocytopenia in mice: dissociation between alterations in megakaryocytes and platelets. J Cell Physiol 1989;141:160–9. 177. Stenberg PE, Levin J, Baker G, Mok Y, Corash L. Neuraminidaseinduced thrombocytopenia in mice: effects on thrombopoiesis. J Cell Physiol 1991;147:7–16. 178. Scholes DR, Paige KN. Plasticity in ploidy underlies plant fitness compensation to herbivore damage. Mol Ecol 2014;23:4862–70. 179. Jackson CW. Cholinesterase as a possible marker for early cells of the megakaryocytic series. Blood 1973;42:413–21. 180. Smith MJ, Braem B, Davis KD. Human platelet acetylcholinesterase: the effects of anticholinesterases on platelet function. Thromb Haemost 1980;42:1615–9. 181. Katakura F, Katzenback BA, Belosevic M. Recombinant goldfish thrombopoietin up-regulates expression of genes involved in thrombocyte development and synergizes with kit ligand A to promote progenitor cell proliferation and colony formation. Dev Comp Immunol 2015;49:157–69.

1