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1958;7(1):45–63.

Part I

Platelet Biology

Chapter 1 The Evolution of Mammalian Platelets

Chapter 2 Megakaryocyte Development and Platelet Formation

Chapter 3 The Regulation of Platelet Life Span

Chapter 4 Platelet Genomics

Chapter 5 Platelet microRNAs

Chapter 6 The Platelet Proteome

Chapter 7 Platelet Structure

Chapter 8 The Platelet Cytoskeleton

Chapter 9 Platelet Receptors

Chapter 10 The GPIb-IX-V Complex

Chapter 11 GPVI and CLEC-2

Chapter 12 Integrin αIIbβ3

Chapter 13 Protease-Activated Receptors

Chapter 14 The Platelet P2 Receptors

Chapter 15 PECAM-1

Chapter 16 Interactions Between Platelets, Leukocytes and the Endothelium

Chapter 17 Inhibition of Platelet Function by the Endothelium

Chapter 18 Platelet Secretion

Chapter 19 Signal Transduction During Platelet Plug Formation

Chapter 20 Platelet Thrombus Formation in Flowing Blood

Chapter 21 Interactions Between Platelets and the Coagulation System

Chapter 22 Platelet-Derived Microparticles

Chapter 23 The Role of Platelets in Fibrinolysis

Chapter 24 The Role of Platelets in Angiogenesis

Chapter 25 Platelet Function in the Newborn

Chapter 1

The Evolution of Mammalian Platelets

Jack Levin

Departments of Laboratory Medicine and Medicine, University of California School of Medicine, San Francisco, California

I 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 cell-based 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, whose functions include hemostasis. For example, platelets possess rudimentary bactericidal and phagocytic activity (see Chapter 37). They have been shown to interact with bacteria, endotoxins, viruses, parasites, and fungi. Platelets are not only important for the maintenance of hemostasis but are also inflammatory cells (see Chapter 36). Despite these overall similarities in function, there exists no proof that invertebrate blood cells evolved into platelets. Furthermore, the presence of nonnucleated platelets and their polyploid megakaryocyte progenitors in the bone marrow 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.

II 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 ethylenediaminetetraacetic 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.⁵ Aggregation in vitro was shown to be a two-step process and distinct from adhesion. Adhesion is Ca++- or Mg++-dependent.⁵ The parallels with mammalian platelet function are evident.

Figure 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 Figures 1-15, 1-17, and 1-18.) Magnification ×200 (upper) and ×320 (lower).

(From Levin and Bang,¹ with permission.)

Figure 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.)

Figure 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 (0 minute) and subsequent collections is indicated above each tracing. The bar represents 5 minutes. The extent of light scattering is shown on the ordinate in arbitrary units.

(From Takahashi et al.,³ with permission.)

The enormous range of types of cell-based coagulation in insects has been described by Grégoire.⁶ The hemolymph of many insects contains coagulocytes, which upon 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 hemolymph of the blowfly Calliphora erythrocephala tend to fragment. Their fragments then agglutinate and form networks that are believed to promote hemostasis and seal wounds.⁷ Notably, each cell fragment remains surrounded by an intact plasma membrane.

Figure 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 Grégoire,⁶ with permission.)

Figure 1-5 Erodius tibialis (Coleoptera).

The threadlike cytoplasmic processes are shown, carrying along the granules produced by six coagulocytes (asterisks). Magnification ×960.

(From Grégoire, ⁶ with permission.)

In some invertebrates, hemostasis is provided entirely by cell aggregation at the site of a wound.⁸–¹⁰ In others, the hemocytes contain coagulation factors or clottable protein that are released after activation and/or aggregation of the cells.¹¹–¹⁴ 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.¹⁵ In some arthropods, circulating amebocytes release a coagulase that activates a clottable protein already in the plasma.¹⁶ Plasma from the hemolymph of L. polyphemus normally lacks coagulation factors.¹²–¹³ 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.¹⁷,¹⁸ Activation of Limulus amebocytes not only results in initiation of coagulation but is also associated with the initiation of other defense mechanisms, including wound healing, activation of a primitive complement system, and release of antibacterial factors.¹⁹

III Nonmammalian Vertebrates

Nonmammalian vertebrates have nucleated, often spindle-shaped thrombocytes, the first cells to evolve that specialize in hemostasis.⁸,¹¹,²⁰ Thrombocytes are found in fish, and in some species multiple types of thrombocytes have been described.²¹,²² 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, variable number of cytoplasmic granules.²³ There is 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. In addition, size differences may exist between immature and mature thrombocytes.²⁴ Quantification of fish thrombocytes has been made difficult by the often-described resemblance between fish thrombocytes and lymphocytes and by the tendency of thrombocytes to clump.²¹ The most extensive quantitative 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.²⁵ 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.²⁶,²⁷ Thrombocytes of the piracanjuba Brycon orbignyanus²⁶ and the Murray cod Maccullochella peelii peelii²⁷ were PAS-positive but negative for a wide range of other cytochemical markers (Fig. 1-6). In contrast, the thrombocytes of seven other species of fish were positive for both PAS and acid phosphatase.²⁸,²⁹ 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 naphthol AS-D chloroacetate esterase (AS-D).³⁰ Examples of thrombocytes in cartilaginous fish are shown in Fig. 1-7.³¹

Figure 1-6 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 Shiff (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=10 μm.

(Modified from Shigdar et al.,²⁷ with permission).

Figure 1-7 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,³¹ with permission.)

Some species of estuarine cyprinodontiform fishes have been described as having a seasonal variation in the ratio of mature to immature circulating thrombocytes.²⁴ 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 microtubules ²⁹,³² and are in at least some species described as phagocytic.²⁹,³³–³⁵ In contrast to platelets, thrombocytes are not typically aggregated by adenosine diphosphate (ADP) or epinephrine.⁸,³⁶,³⁷ Zebrafish (Danio rerio) thrombocytes have been shown in vivo to play a role in the development of arterial thrombi.³⁸ 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.³⁹

Avian thrombocytes are similar in appearance to fish thrombocytes and are believed to be produced by mononuclear precursors in the bone marrow.⁴⁰ Four developmental stages of thrombocyte precursors have been described in the chick embryo and collectively account for 0.6 to 2.4% of nucleated cells in the bone marrow.⁴¹ 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.⁴² Flow cytometry has been used to discriminate between lymphocytes and thrombocytes in chickens⁴³ and ducks.⁴⁴ The developmental stages of the thrombocyte lineage in chicken are shown in Fig. 1-8.⁴⁵ Phagocytosis has been demonstrated.⁴⁶ As in fish, thrombocytes are the most common white blood cell in the chicken⁴³ and in ducks.⁴⁴ Similar to platelets, avian thrombocytes contain serotonin (5-hydroxytryptamine)⁴⁷,⁴⁸ and release what appears to be β-thromboglobulin during the release reaction.⁴⁹

Figure 1-8 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,⁴⁵ with permission.)

The thrombocytes of at least some birds, amphibians, reptiles, and fish have a membrane system referred to as the surface-connected canalicular system (SCCS).³²,⁵⁰–⁵⁴ This system is also a feature of mammalian platelets and has been linked to their derivation from the cytoplasm of megakaryocytes.⁵⁵ 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. Bovine⁵⁶ and African elephant (Loxodonta africana)⁵⁷ platelets are apparently exceptions in that they do not contain a SCCS, although bovine megakaryocytes have a DMS.⁵⁵ Significantly, the platelets of the African elephant also lack microtubules.⁵⁷ 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.⁵⁸ 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. 1-9. Multiple examples of the thrombocytes of other nonmammalian vertebrate species are shown in Fig. 1-10.⁵⁹–⁶³ 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.⁶⁴ However, the thrombocytes of two species of Australian crocodiles were PAS-positive.⁶⁵ Serotonin has been detected immunochemically in seagull, alligator, and sea turtle thrombocytes (Fig. 1-11) and its presence in alligator and sea turtle thrombocytes confirmed with high-performance liquid chromatography (HPLC) (Fig. 1-12).⁴⁸ Serotonin has not been detected in the thrombocytes of the American bullfrog Rana catesbeiana⁴⁸ or the tortoise Geoclemys reevesii.⁶⁶ 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. 1-13).⁴⁸

Figure 1-9 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 (Rana catesbeiana) thrombocytes. Two of the thrombocytes contain large nuclei. The inclusion in the lowest cell may be a phagocytosed red blood cell.

(From Lewis,³¹ with permission.)

Figure 1-10 Thrombocytes of the common lizard ( Podarcis s. sicula Raf.), dogfish ( Scyliorhynus stellaris L.), electric ray ( Torpedo marmorata ), red-legged partridge ( Alectoris rufa rufa L.), and eagle ( Aquila rapax ).

Top panel: A. Group of lizard thrombocytes (May Grünwald Giemsa). B. Lizard thrombocytes positively stained for acid phosphatase. C. Dogfish thrombocyte with 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). [Insert: 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).

(From Pica et al.,⁵⁹–⁶¹ D’Ippolito et al.,⁶² and Jain,⁶³ with permission.)

Figure 1-11 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=10 μm.

(From Maurer-Spurej,⁴⁸ with permission.)

Figure 1-12 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 freshwater turtle, frog, tuna, or salmon do not.

(From Maurer-Spurej,⁴⁸ with permission.)

Figure 1-13 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, 100–1,000, and 1,000–5,000 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,⁴⁸ with permission.)

IV Comparative Hemostasis

An overview of comparative hemostasis is provided in Table 1-1.⁶⁷ 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.⁸,⁶⁸

Table 1-1 Summary of Hemostatic Mechanisms So Far Identified in Various Groups of Animals

Adapted from Hawkey.⁶⁷ See also Needham.⁹

aIn some examples, the term clottable protein would be more appropriate.

+Designate various components or phases of hemostasis in invertebrates which approximate those present and better defined in vertebrate classes.

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 animals (Fig. 1-14).⁶⁹ 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.⁶⁹ (p. 233–234)

Figure 1-14 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 Hewson,⁶⁹ Plate V, p. 312, from Section III [originally published in 1777]; from the author’s collection.)

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. 1-14, an original Hewson plate; compare his figures 11 and 12).

V 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 studied of the invertebrate blood cells involved in hemostasis and blood coagulation.¹,⁷⁰ 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. 1-15).⁷¹ After activation on a foreign surface (or by bacterial endotoxins), amebocytes spread and degranulate (Fig. 1-15, right panel). Degranulation is associated with exocytosis.¹²,⁷²–⁷⁴ The amebocytes, which are normally discoid (like platelets), develop pseudopodia and microspikes.⁷⁵ These cells are also capable of retraction (Fig. 1-16),⁷⁶ a process apparently similar to that of clot retraction induced by mammalian platelets.

Figure 1-15 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 ×1,000.

(From Levin,⁷¹ with permission.)

Figure 1-16 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 Söderhäll et al.,⁷⁶ with permission.)

Fig. 1-17⁷⁷ shows the ultrastructure of a Limulus amebocyte. The cytoplasm contains at least two types of granules (Fig. 1-18)⁷⁸ that are biochemically different and contain all the components of the blood coagulation mechanism.¹⁷,⁷⁰,⁷⁸ Marginal microtubule bands are present (Fig. 1-19).

Figure 1-17 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 ×7,000.

(From Copeland and Levin,⁷⁷ with permission.)

Figure 1-18 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 ×6,140. (From Copeland and Levin, ⁷⁷ with permission.) Right: Scanning electron micrograph of a preparation of intact cytoplasmic granules obtained from Limulus amebocytes. Magnification ×10,000.

(From Mürer et al.,⁷⁸ with permission.)

Figure 1-19 A marginal microtubule band is demonstrated in a cross-section 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 microtubule-associated proteins present in other systems. Magnification ×104,000.

(From Tablin and Levin,⁷⁵ with permission.)

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 recently 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.⁷⁹ The bactericidal mechanism was not established. Despite more than 400 million years of evolution,⁸⁰ 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, whose functions include hemostasis. Studies of the thrombocytes of the rainbow trout Oncorhynchus mykiss concluded that these cells had both hemostatic and phagocytic functions, and the authors speculated that some aspects of this dual functionality observed in thrombocytes may have been lost with the evolution of the anucleate platelet.⁸¹

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.⁷⁹,⁸²–⁸⁵ 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).⁸⁶ Nevertheless, an exhaustive review of studies that described the interactions of thrombocytes and platelets with particulate materials or bacteria presented considerable data, which appeared to support the conclusion that both thrombocytes and platelets were capable of phagocytosis.⁸⁷

Table 1-2 Comparison of Limulus Amebocytes and Mammalian Platelets

Platelets contain endotoxin-binding substances⁸⁸ and have been shown to interact with bacteria, endotoxins, viruses, and fungi.⁸⁹–⁹³ Murine⁹⁴ and human⁹⁵ platelets and chicken thrombocytes⁹⁶ have been shown to express a functional toll-like receptor-4 (TLR4), the receptor for bacterial endotoxin (LPS).⁹⁴ The extensive role of platelets in antimicrobial host defense has been reviewed (see Chapter 37).⁹³,⁹⁷ Staphylococcus can stimulate human platelets to undergo the release reaction.⁹⁸ 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.⁹⁹ 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.¹⁰⁰ 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.¹⁰¹ Certain surface proteins of Streptococcus gordonii bind to the extracellular portion of the platelet membrane glycoprotein (GP)Ibα.¹⁰² Pneumococcus (S. pneumoniae) and vaccinia virus can induce the release of serotonin from human platelets.¹⁰³,¹⁰⁴ Other investigations have demonstrated that two endocarditis-producing strains of S. viridans can activate human platelets in vitro,¹⁰⁵ apparently by direct platelet-bacterial interaction. Fibrinogen in conjunction with other unidentified plasma factors was required for platelet aggregation and secretion.¹⁰⁵,¹⁰⁶ The aggregation of human platelets by S. viridans, S. pyogenes, and S. sanguis has been demonstrated.¹⁰⁷–¹⁰⁹ S. mitis has been shown to possess at least two genomic regions that contribute to platelet binding.¹¹⁰

Human platelets have been reported to be cytotoxic for parasites by a mechanism involving an IgE receptor on the platelet surface.¹¹¹,¹¹² Platelets also have been shown to play a role in the excretion of Schistosoma mansoni in the stool of mice.¹¹³ 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.¹¹⁴ Under certain circumstances, platelets demonstrate a chemotactic response and migrate,¹¹⁵–¹¹⁷ as do amebocytes.¹¹⁸ It has been concluded that the thrombocytes of the turbot Psetta maxima are also capable of movement.²⁹ 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 37 for further details).

VI 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 an ancient mechanism. The limited ability of mammalian platelets to phagocytose (or internalize) particles 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. 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 mechanism¹¹⁹ (see Chapter 37).

Quick¹²⁰ stated: 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,¹²¹ referring to the evolution of blood coagulation and hemostasis, wrote of man, who must realize that the imprints of a very distant past are still with him (p. 305). Insightfully, Quick¹²⁰ 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). Needham⁹ has articulated the same hypothesis. In summary, platelets are not only important for the maintenance of hemostasis but are also inflammatory cells (see Chapter 36) that have important roles in antimicrobial host defense mechanisms⁹³,⁹⁷,¹²²,¹²³ (see Chapter 37).

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—for example, 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 Quick,¹²⁰

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)

Some evolutionary aspects of blood coagulation have been reviewed.⁸,¹⁶,¹⁷,³⁴,¹²⁴ Strikingly, basal chordates (protochordates) do not appear to have a plasma-based coagulation mechanism.¹²⁴ 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, which seems to be a marked departure from all other groups of animals, even taking into account the evolutionary concept of punctuated equilibria.¹²⁵,¹²⁶ However, as thoughtfully stated by Ratcliffe and Millar,¹²⁷ 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.¹²⁸–¹³⁰ These studies have been supported by the observation that the aggregation of pigeon thrombocytes by thrombin in the presence of calcium and fibrinogen was inhibited by an anti-GPIIb antibody.¹³¹ GPIIb-IIIa (integrin αIIbβ3) has also been detected on thromboblasts in chick embryos and, notably, the level of its expression was related to the differentiation of thromboblasts (which were colony-forming cells) into thrombocytes (not colony-forming cells).¹³⁰ The previously mentioned studies have been recently supported by the application of quantitative polymerase chain reaction (PCR) and microarray analysis, which detected genes which encoded for the Mpl receptor, the α2 and β3 integrins, and the GPIb-V-IX receptor complex on chicken thrombocytes.³⁹ Analogs of the GPIIb-IIIa 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.¹³² Zebrafish thrombocytes also have been demonstrated to have platelet GPIb and GPIIb-IIIa-like complexes on their surfaces.¹³³ In addition, zebrafish thrombocyte precursors are c-mpl-positive, and injection of an antisense c-mpl morpholino into embryos eliminated the production of thrombocytes.¹³⁴

VII Megakaryocytes and Mammals

Nonnucleated platelets, and presumably their polyploid megakaryocyte progenitors in the bone marrow (and also in the spleen in some animals), are present only in mammals, which suggests that some important feature of mammalian physiology benefits from this unique mechanism for producing an unprecedented 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, 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 fluids, and most (nonmammalian vertebrates being particularly relevant) have effective hemostatic mechanisms, none of which depend upon a megakaryocyte/platelet axis. We must ask, therefore, what the biological advantage is 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.

A Marine Mammals

Marine mammals are unique because, with the exception of the duckbilled platypus, they 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.¹³⁵ However, their platelets are morphologically similar to other mammalian platelets (Fig. 1-20). 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.¹³⁶ Only limited data are available for platelet counts in marine mammals.¹³⁷–¹⁴² 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).

Figure 1-20 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.,¹³⁵ with permission.)

Table 1-3 Platelet Counts in Selected Marine Mammals

n.a.=not available.

Data from Reidarson et al.¹⁴² (Almost all the published platelet counts of marine mammals have been provided by the clinical laboratory of Sea World.)

aThe order (subclass) within the class Mammalia is shown.

bMean platelet count. The number of animals studied is shown in parentheses.

B 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 on echidna platelets¹⁴³ 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). Hawkey¹⁴³ 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 study¹⁴⁴ 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/L¹⁴⁵ and 205 to 682×10⁹/L (18 animals).¹⁴⁶

Platelet levels of approximately 400 to 450×10⁹/L were reported for the duckbilled platypus Ornithorhynchus anatinus.¹⁴⁷ The same group of investigators¹⁴⁸ 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.¹⁴⁸ 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 Hawkey¹⁴³ 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.¹⁴⁶ 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.¹⁴⁹ 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.

C Order Marsupialia

Marsupials—represented by the opossum, kangaroo, wombat, and bandicoot—give birth to live but very immature young after a nonplacental pregnancy. 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.¹⁵⁰ 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. 1-21. For comparison, electron micrographs of platelets in other mammalian orders are shown in Fig. 1-22. In other investigations, platelet levels in five different species of marsupials ranged from approximately 200 to 500×10⁹/L,¹⁴³,¹⁴⁴,¹⁵¹,¹⁵² 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 wallaby¹⁵³ (Table 1-4). Release of serotonin, a characteristic of mammalian platelets, was also demonstrated for marsupial platelets.¹⁵¹ In two Australian marsupial species whose bone marrow has been studied, megakaryocytes were described but not illustrated.¹⁴⁴ Microscopy studies of the liver of the North American opossum Didelphis virginiana revealed the presence of significant numbers of megakaryocytes in pouch-young animals.¹⁵⁴ (These animals remain attached to the nipple for at least 60 days and leave the pouch only afterward.) 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.¹⁵⁴ The number of megakaryocytes in the liver decreased with age.¹⁵⁴,¹⁵⁵ As the animals increased in age, mature megakaryocytes appeared, and extensive demarcation membranes became clearly apparent. As is the pattern for mammalian megakaryocytes, maturation of the nucleus generally preceded cytoplasmic maturation.¹⁵⁵ Concomitantly, megakaryocytopoiesis occurred in the hepatic sinusoids. Neither paper described the bone marrow or spleen as playing a role in the production of platelets. Notably, 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.¹⁵⁶ Fig. 1-23 presents micrographs of the bone marrow of the South American opossum (Monodelphis domestica), which provide multiple examples of megakaryocytes in this marsupial.

Figure 1-21 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,³¹ with permission.)

Figure 1-22 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. Right: 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. Bottom: 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,³¹ with permission.)

Table 1-4 Platelet Counts in Selected Mammals

Data were derived from Lewis.³¹

aThe order (subclass) within the class Mammalia is shown.

bMean platelet count. The number of animals studied is shown in parentheses.

Figure 1-23 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.

D Platelet Levels

It is interesting that 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.¹⁵⁷ On the basis of the previously published nomogram and platelet levels in some additional mammals, 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.¹⁵⁸–¹⁶⁰ Mean platelet volume is not related to body mass.¹⁵⁹–¹⁶¹

E 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, has also been observed in both orders.¹⁴⁴,¹⁵¹ 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.¹⁶² Nevertheless, a potential role for platelets during pregnancy in mammals has been described.¹⁶³ 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.¹⁶³ 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.

VIII Conclusions

There is presumably a biological advantage gained from the presence of polyploid megakaryocytes as the source for nonnucleated platelet progeny in mammals, but 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.¹⁶⁴ In addition, cell growth and differentiation can continue, uninterrupted by nuclear and cell divisions. Nagl¹⁶⁴ 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 platelets have not yet 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.¹⁶⁵,¹⁶⁶ 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.

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.¹⁶⁷ 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.¹⁶⁸

The 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, remain elusive. 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. 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 almost 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 MBL who provided me with the information and techniques necessary 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.

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

Supported in part by the Veterans Administration.

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