Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 13. Platelet production, structure, and function

George A. Fritsma



Megakaryocyte Differentiation and Progenitors


Terminal Megakaryocyte Differentiation

Megakaryocyte Membrane Receptors and Markers

Thrombocytopoiesis (Platelet Shedding)

Hormones and Cytokines of Megakaryocytopoiesis


Platelet Ultrastructure

Resting Platelet Plasma Membrane

Surface-Connected Canalicular System

Dense Tubular System

Platelet Plasma Membrane Receptors That Provide for Adhesion

The Seven-Transmembrane Receptors

Additional Platelet Membrane Receptors

Platelet Cytoskeleton: Microfilaments and Microtubules

Platelet Granules: a-Granules, Dense Granules, and Lysosomes

Platelet Activation

Adhesion: Platelets Bind Elements of the Vascular Matrix

Aggregation: Platelets Irreversibly Cohere

Secretion: Activated Platelets Release Granular Contents

Platelet Activation Pathways

G Proteins

Eicosanoid Synthesis

Inositol Triphosphate Diacylglycerol Activation Pathway


After completion of this chapter, the reader will be able to:

1. Diagram megakaryocyte localization in bone marrow.

2. List the transcription products that trigger and control megakaryocytopoiesis and endomitosis.

3. Diagram terminal megakaryocyte differentiation, the proplatelet process, and thrombocytopoiesis.

4. Describe the ultrastructure of resting platelets in the circulation, including the plasma membrane, tubules, microfibrils, and granules.

5. List the important platelet receptors and their ligands.

6. Recount platelet function, including adhesion, aggregation, and secretion.

7. Reproduce the biochemical pathways of platelet activation, including integrins, G proteins, the eicosanoid, and the diacylglycerol-inositol triphosphate pathway.


After studying this chapter, the reader should be able to respond to the following case study:

A 35-year-old woman noticed multiple pinpoint red spots and bruises on her arms and legs. The hematologist confirmed the presence of petechiae, purpura, and ecchymoses on her extremities and ordered a complete blood count, prothrombin time, and partial thromboplastin time. The platelet count was 35 × 109/L, the mean platelet volume was 13.2 fL, and the diameter of platelets on the Wright-stained peripheral blood film appeared to exceed 6 μm. Other complete blood count parameters and the coagulation parameters were within normal limits. A Wright-stained bone marrow aspirate smear revealed 10 to 12 small unlobulated megakaryocytes per low-power microscopic field.

1. Do these signs and symptoms indicate systemic (mucocutaneous) or anatomical (soft tissue) bleeding?

2. What is the probable cause of the bleeding?

3. Is the thrombocytopenia the result of inadequate bone marrow production?

4. List the growth factors involved in recruiting megakaryocyte progenitors.


Platelets are nonnucleated blood cells that circulate at 150 to 400 × 109/L, with average platelet counts slightly higher in women than in men and slightly lower in both sexes when over 65 years old.1 Platelets trigger primary hemostasis upon exposure to subendothelial collagen or endothelial cell inflammatory proteins at the time of blood vessel injury. On a Wright-stained wedge-preparation blood film, platelets are distributed throughout the red blood cell monolayer at 7 to 21 cells per 100× field. On the blood film they have an average diameter of 2.5 μm, corresponding to a mean platelet volume (MPV) of 8 to 10 fL when measured by impedance in a buffered isotonic suspension, as determined using laboratory profiling instruments.2 Their internal structure, although complex, is granular but scarcely visible using light microscopy.

Platelets arise from unique bone marrow cells called megakaryocytes. Megakaryocytes are the largest cells in the bone marrow and are polyploid, possessing multiple chromosome copies. On a Wright-stained bone marrow aspirate smear, each megakaryocyte is 30 to 50 μm in diameter with a multilobulated nucleus and abundant granular cytoplasm. Megakaryocytes account for less than 0.5% of all bone marrow cells, and on a normal Wright-stained bone marrow aspirate smear the microscopist may identify two to four megakaryocytes per 10× low-power field (Chapter 7).3

In healthy intact bone marrow tissue, megakaryocytes, under the influence of an array of stromal cell cytokines, cluster with hematopoietic stem cells in vascular niches adjacent to venous sinusoid endothelial cells ().Figure 13-14 Responding to the growth factor thrombopoietin (TPO), megakaryocyte progenitors are recruited from common myeloid progenitors (Chapter 7) and subsequently differentiate through several maturation stages. They extend proplatelet processes, projections that resemble strings of beads, through or between the endothelial cells and into the venous sinuses, releasing platelets from the tips of the processes into the circulation. Megakaryocytes are also found in the lungs.5


FIGURE 13-1 Cross section of bone marrow hematopoietic tissue. The nerve, artery, and vein run longitudinally through the center of the marrow. Venous sinuses extend laterally from the central vein throughout the hematopoietic tissue. Differentiating and mature megakaryocytes localize to the abluminal (non-blood) surface of sinusoid-lining endothelial cells.

Megakaryocyte differentiation and progenitors

Megakaryocyte progenitors arise from the common myeloid progenitor under the influence of the transcription gene product, GATA-1, regulated by cofactor FOG1 ().Box 13-14 Megakaryocyte differentiation is suppressed by another transcription gene product, MYB, so GATA-1 and MYB act in opposition to balance megakaryocytopoiesis in one arm with differentiation to the red blood cell line in another arm, called erythropoiesis. From the common myeloid progenitor there arise three megakaryocyte lineage-committed progenitor stages, defined by their in vitro culture colony characteristics (Figure 7-13). In order of differentiation, these are the least mature burst-forming unit (BFU-Meg), the intermediate colony-forming unit (CFU-Meg), and the more mature progenitor, the light-density CFU (LD-CFU-Meg).6 All three progenitor stages resemble lymphocytes and cannot be distinguished by Wright-stained light microscopy. The BFU-Meg and CFU-Meg are diploid and participate in normal mitosis, maintaining a viable pool of megakaryocyte progenitors. Their proliferative properties are reflected in their ability to form hundreds (BFU-Megs) or scores (CFU-Megs) of colonies in culture (Figure 13-2).7 The third stage, LD-CFU-Meg, loses its capacity to divide but retains its DNA replication and cytoplasmic maturation, a partially characterized form of mitosis unique to megakaryocytes known as endomitosis.


FIGURE 13-2 Three stages of megakaryocyte progenitors. The BFU-Meg clones hundreds of daughter cells, the CFU-Meg clones scores of daughter cells, and the LD-CFU-Meg undergoes the first stage of endomitosis. BFU-Meg, Burst-forming unit megakaryocyte; CFU-Meg, colony-forming unit megakaryocyte; CFU-GEMM, colony-forming unit, granulocyte, erythrocyte, monocyte, megakaryocyte, LD-CFU-Meg, low-density CFU-Meg; TPO, thrombopoietin; Meg-CSF, megakaryocyte colony stimulating factor; IL-3, interleukin-3.

BOX 13-1


Endomitosis is no longer a mystery to the molecular biologists who mapped its translational control genes, but it may seem mysterious to those of us who do not work daily with gene, gene product, and mutation abbreviations. The abbreviations have antecedents—for instance, GATA-1 stands for “globin transcription factor-1,” a protein product of the X chromosome gene GATA1. FOG1 stands for “friend of GATA,” a product of the ZFPM1 (zinc finger protein multitype 1) gene. Laboratory scientists rarely refer to gene names, only their abbreviations, though they are familiar with their characteristics and functions. The abbreviations have moved rapidly into clinical laboratory jargon, as they are becoming key diagnostic markers. In this chapter we use gene product abbreviations without reference to their antecedents.


Endomitosis is a form of mitosis that lacks telophase and cytokinesis (separation into daughter cells). As GATA-1 and FOG1 transcription slows, another transcription factor, RUNX1, mediates the switch from mitosis to endomitosis by suppressing the Rho/ROCK signaling pathway, which suppresses the assembly of the actin cytoskeleton.8 In response to the reduced Rho/ROCK signal, inadequate levels of actin and myosin (muscle fiber–like molecules) assemble in the cytoplasmic constrictions where separation would otherwise occur, preventing cytokinesis. Subsequently, under the influence of yet another transcription factor, NF-E2, DNA replication proceeds to the production of 8N, 16N, or even 32N ploidy with duplicated chromosome sets.4 Some megakaryocyte nuclei replicate five times, reaching 128N; this level of ploidy is unusual, however, and may signal hematologic disease.

Megakaryocytes employ their multiple DNA copies to synthesize abundant cytoplasm, which differentiates into platelets. A single megakaryocyte may shed 2000 to 4000 platelets, a process called thrombopoiesis or thrombocytopoiesis. In an average-size healthy human there are 108 megakaryocytes producing 1011 platelets per day, a total turnover rate of 8 to 9 days. In instances of high platelet consumption, such as immune thrombocytopenic purpura, platelet production rises by as much as tenfold.

Terminal megakaryocyte differentiation

As endomitosis proceeds, megakaryocyte progenitors leave the proliferative phase and enter terminal differentiation, a series of stages in which microscopists become able to recognize their unique Wright-stained morphology in bone marrow aspirate films (Figure 13-3) or hematoxylin and eosin–stained bone marrow biopsy sections (Table 13-1).


FIGURE 13-3 Morphologically identifiable megakaryocytes of the terminal megakaryocyte differentiation compartment. LD-CFU-Meg, Low-density colony forming unit megakaryocyte; MK-I, megakaryocyte I or megakaryoblast; MK-II, megakaryocyte II or promegakaryocyte; MK-III, megakaryocyte III or megakaryocyte; TPO, thrombopoietin; IL-11, interleukin 11.

TABLE 13-1

Features of the Three Terminal Megakaryocyte Differentiation Stages





% of precursors





14—18 μm

15—40 μm

30—50 μm








Not visible



Moderately condensed

Deeply and variably condensed

Nucleus-to-cytoplasm ratio














Basophilic and granular

Azurophilic and granular





Dense granules




Demarcation system




MK-I, Megakaryoblast; MK-II, promegakaryocyte; MK-III, megakaryocyte.

Morphologists call the least differentiated megakaryocyte precursor the MK-I stage or megakaryoblast. Although they no longer look like lymphocytes, megakaryoblasts cannot be reliably distinguished from bone marrow myeloblasts or pronormoblasts (also named rubriblasts) using light microscopy (Figure 13-4). The morphologist may occasionally see a vague clue: plasma membrane blebs, blunt projections from the margin that resemble platelets. The megakaryoblast begins to develop most of its cytoplasmic ultrastructure, including procoagulant-laden α-granules, dense granules (dense bodies), and the demarcation system (DMS).9


FIGURE 13-4 The megakaryoblast (MK-I) resembles the myeloblast and pronormoblast (rubriblasts); identification by morphology alone is inadvisable. This megakaryoblast has cytoplasmic “blebs” that resemble platelets.

The contents and functions of α-granules and dense granules are described in the subsequent sections on mature platelet ultrastructure and function. The DMS is a series of membrane-lined channels that invade from the plasma membrane and grow inward to subdivide the entire cytoplasm. The DMS is biologically identical to the megakaryocyte plasma membrane and ultimately delineates the individual platelets during thrombocytopoiesis.

Nuclear lobularity first becomes apparent as an indentation at the 4N replication stage, rendering the cell identifiable as an MK-II stage, or promegakaryocyte, by light microscopy. The morphologist seldom makes the effort to distinguish MK-I, MK-II, and MK-III stages during a routine examination of a bone marrow aspirate smear.

The promegakaryocyte reaches its full ploidy level by the end of the MK-II stage. At the most abundant MK-III stage, the megakaryocyte is easily recognized at 10× magnification on the basis of its 30- to 50-μm diameter (Figures 13-5 and 13-6). The nucleus is intensely indented or lobulated, and the degree of lobulation is imprecisely proportional to ploidy. When necessary, ploidy levels are measured using mepacrine, a nucleic acid dye in megakaryocyte flow cytometry.10 The chromatin is variably condensed with light and dark patches. The cytoplasm is azurophilic (lavender), granular, and platelet-like because of the spread of the DMS and α-granules. At full maturation, platelet shedding, or thrombocytopoiesis, proceeds.


FIGURE 13-5 Promegakaryocyte (MK II). Cytoplasm is abundant, and nucleus shows minimal lobularity.


FIGURE 13-6 Megakaryocyte. The nucleus is lobulated with basophilic chromatin. The cytoplasm is azurophilic and granular, with evidence of the demarcation system (DMS).

Megakaryocyte membrane receptors and markers

In specialty and tertiary care laboratories, scientists and technicians employ immunostaining of fixed tissue, flow cytometry with immunologic probes, and fluorescent in situ hybridization (FISH) with genetic probes to identify visually indistinguishable megakaryocyte progenitors in hematologic disease. There are several flow cytometric megakaryocyte membrane markers, including MPL, which is the TPO receptor site present at all maturation stages, and the stem cell and common myeloid progenitor marker CD34. The CD34 marker disappears as differentiation proceeds. The platelet membrane glycoprotein IIb/IIIa (CD41, a marker located on the IIb portion) first appears on megakaryocyte progenitors and remains present throughout maturation, along with immunologic markers CD36, CD42, CD61, and CD62. Cytoplasmic coagulation factor VIII, von Willebrand factor (VWF), and fibrinogen, may be detected by immunostaining in the fully developed megakaryocyte ().Table 13-211-13

TABLE 13-2

Markers at Each Stage of Megakaryocyte Maturation Detected by Flow Cytometry, Immunostaining, Fluorescence In Situ Hybridization, or Cytochemical Stain









MPL, TPO receptor by FCM


CD34; stem cell marker by FCM


CD41: β3 portion of αIIbβ3; peroxidase by TEM cytochemical stain


CD42; GP Ib portion of VWF receptor, by FCM


PF4 by FCM


VWF by immunostaining




The arrows indicate at which stage of differentiation the marker appears and at which stage it disappears. BFU-Meg, Burst-forming unit–megakaryocyte; CFU-Meg, colony-forming unit–megakaryocyte; LD-CFU-Meg, low-density colony-forming unit–megakaryocyte; MK-I,megakaryoblast; MK-II, promegakaryocyte; MK-III, megakaryocyte; FCM, flow cytometry; TEM, transmission electron microscopy; GP, glycoprotein; PF4, platelet factor 4; TPO, thrombopoietin; VWF, von Willebrand factor

Thrombocytopoiesis (platelet shedding)

shows platelet shedding, termed Figure 13-7 thrombocytopoiesis. One cannot find reliable evidence for platelet budding or shedding simply by examining megakaryocytes in situ, even in well-structured bone marrow biopsy preparations. However, in megakaryocyte cultures examined by transmission electron microscopy, the DMS dilates, longitudinal bundles of tubules form, proplatelet processes develop, and transverse constrictions appear throughout the proplatelet processes. In the bone marrow environment, processes are believed to pierce through or between sinusoid-lining endothelial cells, extend into the venous blood, and shed platelets (Figure 13-8). Thrombocytopoiesis leaves behind naked megakaryocyte nuclei to be consumed by marrow macrophages.14


FIGURE 13-7 This image illustrates a terminal megakaryocyte shedding platelets.


FIGURE 13-8 Megakaryocyte is adjacent to the abluminal (nonblood) membrane of the sinusoid-lining endothelial cell and extends a proplatelet process through or between the endothelial cells into the vascular sinus. Source: (Modified from Powers LW: Diagnostic hematology: clinical and technical principles, St. Louis, 1989, Mosby.)

Hormones and cytokines of megakaryocytopoiesis

The growth factor TPO is a 70,000 Dalton molecule that possesses 23% homology with the red blood cell–producing hormone erythropoietin ().Table 13-315 Messenger ribonucleic acid (mRNA) for TPO has been found in the kidney, liver, stromal cells, and smooth muscle cells, though the liver has the most copies and is considered the primary source. TPO circulates as a hormone in plasma and is the ligand that binds the megakaryocyte and platelet membrane receptor protein identified above, MPL, named for v-mpl, a viral oncogene associated with murine myeloproliferative leukemia. The plasma concentration of TPO is inversely proportional to platelet and megakaryocyte mass, implying that membrane binding and consequent removal of TPO by platelets is the primary platelet count control mechanism.16 Investigators have used both in vitro and in vivo experiments to show that TPO, in synergy with other cytokines, induces stem cells to differentiate into megakaryocyte progenitors and that it further induces the differentiation of megakaryocyte progenitors into megakaryoblasts and megakaryocytes. TPO also induces the proliferation and maturation of megakaryocytes and induces thrombocytopoiesis, or platelet release (Table 13-3). Synthetic TPO mimetics (analogues) elevate the platelet count in patients being treated for a variety of cancers, including acute leukemia. One commercial MPL receptor agonist, romiplostim (NPlate™, Amgen Inc., Thousand Oaks, CA, FDA cleared in 2008), is a nonimmunogenic oligopeptide that is also effective in raising the platelet count in immune thrombocytopenic purpura.17 A second nonpeptide MPL receptor agonist, eltrombopag (Promacta® and Revolade®, Glaxo Smith Kline, Inc., Philadelphia, PA, FDA cleared in 2011), binds and activates an MPL site separate from romiplostim. They may have additive effects.18

TABLE 13-3

Hormones and Cytokines That Control Megakaryocytopoiesis

Cytokine/ Hormone

Differentiation to Progenitors

Differentiation to Megakaryocytes

Late Maturation


Clinical Use






















Cytokines and hormones that have been shown to interact synergistically with TPO and IL-3 include IL-6 and IL-11; stem cell factor, also called kit ligand or mast cell growth factor; granulocyte-macrophage colony–stimulating factor; granulocyte colony–stimulating factor; and erythropoietin.Substances that inhibit megakaryocyte production include platelet factor 4, β-thromboglobulin, neutrophil-activating peptide 2, and IL-8.IL, Interleukin; TPO, thrombopoietin.

Other cytokines that function with TPO to stimulate megakaryocytopoiesis include interleukin-3 (IL-3), IL-6, and IL-11. IL-3 seems to act in synergy with TPO to induce the early differentiation of stem cells, whereas IL-6 and IL-11 act in the presence of TPO to enhance the later phenomena of endomitosis, megakaryocyte maturation, and thrombocytopoiesis. An IL-11 polypeptide mimetic, oprelvekin (Neumega®, Wyeth Ayerst Genetics Institute, Cambridge, MA, FDA cleared in 1997), stimulates platelet production in patients with chemotherapy-induced thrombocytopenia.19 Other cytokines and hormones that participate synergistically with TPO and the interleukins are stem cell factor, also called kit ligand or mast cell growth factor; granulocyte-macrophage colony-stimulating factor (GM-CSF); granulocyte colony-stimulating factor (G-CSF); and acetylcholinesterase-derived megakaryocyte growth stimulating peptide. The list continues to grow.20

Platelet factor 4 (PF4), β-thromboglobulin, neutrophil-activating peptide 2, IL-8, and other factors inhibit in vitro megakaryocyte growth, which indicates that they may have a role in the control of megakaryocytopoiesis in vivo. Internally, reduction in the transcription factors FOG1, GATA-1, and NF-E2 diminish megakaryocytopoiesis at the progenitor, endomitosis, and terminal maturation phases.21


The proplatelet process sheds platelets, cells consisting of granular cytoplasm with a membrane but no nuclear material, into the venous sinus of the bone marrow. Their diameter in the monolayer of a Wright-stained peripheral blood wedge film averages 2.5 μm. MPV, as measured in a buffered isotonic suspension flowing through the impedance-based detector cell of a clinical profiling instrument, ranges from 8 to 10 fL (Figure 1-1). A frequency distribution of platelet volume is log-normal, however, which indicates a subpopulation of large platelets (Figure 15-14). Heterogeneity in the MPV of normal healthy humans reflects random variation in platelet release volume and is not a function of platelet age or vitality, as many authors claim.22

Circulating, resting platelets are biconvex, although the platelets in blood collected using the anticoagulant ethylenediaminetetraacetic acid (EDTA, lavender closure tubes) tend to “round up.” On a Wright-stained wedge-preparation blood film, platelets appear circular to irregular, lavender, and granular, although their small size makes them hard to examine for internal structure.23 In the blood, their surface is even, and they flow smoothly through veins, arteries, and capillaries. In contrast to leukocytes, which tend to roll along the vascular endothelium, platelets cluster with the erythrocytes near the center of the blood vessel. Unlike erythrocytes, however, platelets move back and forth with the leukocytes from venules into the white pulp of the spleen, where both become sequestered in dynamic equilibrium.

The normal peripheral blood platelet count is 150 to 400 × 109/L. The count decreases after 65 years old to 122 to 350 × 109/L in men and 140 to 379 × 109/L in women. This count represents only two thirds of available platelets because the spleen sequesters an additional one third. Sequestered platelets are immediately available in times of demand—for example, in acute inflammation or after an injury, after major surgery, or during plateletpheresis. In hypersplenism or splenomegaly, increased sequestration may cause a relative thrombocytopenia. Under conditions of hemostatic need, platelets answer cellular and humoral stimuli by becoming irregular and sticky, extending pseudopods, and adhering to neighboring structures or aggregating with one another.

Reticulated platelets, sometimes known as stress platelets, appear in compensation for thrombocytopenia (Figure 13-9).22 Reticulated platelets are markedly larger than ordinary mature circulating platelets; their diameter in peripheral blood films exceeds 6 μm, and their MPV reaches 12 to 14 fL.23 Like ordinary platelets, they round up in EDTA, but in citrated (blue-closure tubes) whole blood, reticulated platelets are cylindrical and beaded, resembling fragments of megakaryocyte proplatelet processes. Reticulated platelets carry free ribosomes and fragments of rough endoplasmic reticulum, analogous to red blood cell reticulocytes, which triggers speculation that they arise from early and rapid proplatelet extension and release. Nucleic acid dyes such as thiazole orange bind the RNA of the endoplasmic reticulum. This property is exploited by profiling instruments to provide a quantitative evaluation of reticulated platelet production under stress, a measurement that may be more useful than the MPV.24 Platelet dense granules, however, may interfere with this measurement, falsely raising the reticulated platelet count by taking up nucleic acid dyes. Reticulated platelets are potentially prothrombotic, and may be associated with increased risk of cardiovascular disease.25_29


FIGURE 13-9 A “stress” or “reticulated” platelet. The stress platelet may appear in compensation for thrombocytopenia, which produces early and rapid proplatelet extension and release. The diameter of reticulated platelets exceeds 6 μm. Reticulated platelets carry free ribosomes and fragments of rough endoplasmic reticulum, detectable in flow cytometry using nucleic acid dyes.

Platelet ultrastructure

Platelets, although anucleate, are strikingly complex and are metabolically active. Their ultrastructure has been studied using scanning and transmission electron microscopy, flow cytometry, and molecular sequencing.

Resting platelet plasma membrane

The platelet plasma membrane resembles any biological membrane: a bilayer composed of proteins and lipids, as diagrammed in Figure 13-10. The predominant lipids are phospholipids, which form the basic structure, and cholesterol, which distributes asymmetrically throughout the phospholipids. The phospholipids form a bilayer with their polar heads oriented toward aqueous environments—toward the plasma externally and the cytoplasm internally. Their fatty acid chains, esterified to carbons 1 and 2 of the phospholipid triglyceride backbone, orient toward each other, perpendicular to the plane of the membrane, to form a hydrophobic barrier sandwiched within the hydrophilic layers.


FIGURE 13-10 The platelet possesses a standard biological membrane composed of a phospholipid bilayer with polar head groups oriented toward the aqueous plasma and cytoplasm and nonpolar fatty acid tails that orient toward the center. The phospholipid backbone is interspersed with esterified cholesterol. A series of transmembranous proteins communicate with microfilaments, G proteins, and enzymes. The transmembranous proteins support carbohydrate side chains that extend into the plasma.

The neutral phospholipids phosphatidylcholine and sphingomyelin predominate in the plasma layer; the anionic or polar phospholipids phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine predominate in the inner, cytoplasmic layer. These phospholipids, especially phosphatidylinositol, support platelet activation by supplying arachidonic acid, an unsaturated fatty acid that becomes converted to the eicosanoids prostaglandin and thromboxane A2 during platelet activation. Phosphatidylserine flips to the outer surface upon activation and is the charged phospholipid surface on which the coagulation enzymes, especially coagulation factor complex VIII and IX and coagulation factor complex X and V, assemble.3031

Esterified cholesterol moves freely throughout the hydrophobic internal layer, exchanging with unesterified cholesterol from the surrounding plasma. Cholesterol stabilizes the membrane, maintains fluidity, and helps control the transmembranous passage of materials.

Anchored within the membrane are glycoproteins and proteoglycans; these support surface glycosaminoglycans, oligosaccharides, and glycolipids. The platelet membrane surface, called the glycocalyx, also absorbs albumin, fibrinogen, and other plasma proteins, in many instances transporting them to storage organelles within using a process called endocytosis.

At 20 to 30 nm, the platelet glycocalyx is thicker than the analogous surface layer of leukocytes or erythrocytes. This thick layer is adhesive and responds readily to hemostatic demands. The platelet carries its functional environment with it, meanwhile maintaining a negative surface charge that repels other platelets, other blood cells, and the endothelial cells that line the blood vessels.

The plasma membrane is selectively permeable, and the membrane bilayer provides phospholipids that support platelet activation internally and plasma coagulation externally. The anchored glycoproteins support essential plasma surface–oriented glycosylated receptors that respond to cellular and humoral stimuli, called ligands or agonists, transmitting their stimulus through the membrane to internal activation organelles.

Surface-connected canalicular system

The plasma membrane invades the platelet interior, producing its unique surface-connected canalicular system (SCCS; and Figures 13-1113-12). The SCCS twists spongelike throughout the platelet, enabling the platelet to store additional quantities of the same hemostatic proteins found on the glycocalyx and raising its capacity manyfold. The SCCS also allows for enhanced interaction of the platelet with its environment, increasing access to the platelet interior as well as increasing egress of platelet release products. The glycocalyx is less developed in the SCCS and lacks some of the glycoprotein receptors present on the platelet surface. However, the SCCS is the route for endocytosis and for secretion of α-granule contents upon platelet activation.


FIGURE 13-11 Circulating nonactivated platelet illustrating membrane receptors and activation pathways. The STR receptors include the serotonin receptor 5HT-2A, TXA2 receptor TP, thrombin receptors PAR1 and PAR4, ADP receptors P2Y1 and P2Y12, prostaglandin E2 receptor EP3, and epinephrine receptor, α2. Integrins include collagen receptor β1α2 and fibrinogen/VWF receptor β3αIIb. The key collagen receptor is GPVI, and the key VWF receptor is GP Ib/IX/V. The STR receptors are coupled to G-proteins, which become stimulated when agonists bind their respective receptors to subsequently activate enzymes PLCβ, PI3K, and AC. The activated enzymes in turn activate the PIP2 pathway to produce IP3 and DAG and the eicosanoid synthesis pathway (not shown) to produce TXA2 and cAMP. The integrins and GPs activate PLCγ2 that also activates the PIP2 pathway. These generate shape change, secretion, and aggregation. DTS, Dense tubular system; SCCS, surface-connected canalicular system; TF, tissue factor; TXA2, thromboxane A2TP, TXA2 receptor; PAR1 and PAR4, protease activated receptors that are activated by thrombin; GPVI, glycoprotein VI collagen receptor; GP Ib/IX/V, complex of glycoproteins Ib, IX, and V, which is the VWF receptor; VWF; von Willebrand factor; PLCγ2, phospholipase Cγ2; PLCβ, phospholipase CβPI3K, phosphoinositide-3-kinase; AC, adenyl cyclase; PIP2, phosphotidylinositol-4-5 bisphosphate; PIP3, phosphotidylinositol-4-5 triphosphate; IP3, inositol-1-4-5-triphosphate; DAG, diacyl glycerol.


FIGURE 13-12 Transmission electron micrograph of a circulating nonactivated platelet. Visible are α-granules, vacuoles, and fragments of the surface-connected canalicular system.

Dense tubular system

Parallel and closely aligned to the SCCS is the dense tubular system (DTS), a condensed remnant of the rough endoplasmic reticulum (Figures 13-11 and 13-12). Having abandoned its usual protein production function upon platelet release, the DTS sequesters Ca2+ and bears a series of enzymes that support platelet activation. These enzymes include phospholipase A2, cyclooxygenase, and thromboxane synthetase, which support the eicosanoid synthesis pathway that produces thromboxane A2, and phospholipase C, which supports production of inositol triphosphate (IP3) and diacylglycerol (DAG). The DTS is the “control center” for platelet activation.

Platelet plasma membrane receptors that provide for adhesion

The platelet membrane supports more than 50 categories of receptors, including members of the cell adhesion molecule (CAM) integrin family, the CAM leucine-rich repeat family, the CAM immunoglobulin genefamily, the CAM selectin family, the seven-transmembrane receptor (STR) family, and some miscellaneous receptors.32 Table 13-4 lists the receptors that support the initial phases of platelet adhesion and aggregation.

TABLE 13-4

Glycoprotein Platelet Membrane Receptors That Participate in Adhesion and the Initiation of Aggregation by Binding Specific Ligands

Electrophoresis Nomenclature

Current Nomenclature


Cluster Designation



Integrin: α2β1


CD29, CD49b

Avidity is upregulated via “inside-out” activation that depends on collagen binding to GP VI.


Integrin: αvβ1



Integrin: α5β1


CD29, CD49e


Integrin: α6β1


CD29, CD49f



CAM of the immunoglobulin gene family



Key collagen receptor, triggers activation, release of agonists that increase the avidity of integrins α2β1and αIIbβ3.


CAM of the leucine-rich repeat family

VWF and thrombin bind GP Ibα; thrombin cleaves a site on GP V

CD42a, CD42b, CD42c, CD42d

GP Ib/IX/V is a 2:2:2:1 complex of GP Ibα and Ibβ, GP IX, and GP V. There are 25,000 copies on the resting platelet membrane surface, 5% to 10% on the α-granule membrane, but few on the SCCS membrane. GP Ibα is the VWF-specific site. Fifty percent of GP Ibα/Ibβ is cleared from the membrane on activation. Bernard-Soulier syndrome mutations are identified for all but GP V. Bound to subsurface actin-binding protein.


Integrin: αIIbβ3

Fibrinogen, VWF

CD41, CD61

GP IIb and IIIa are distributed on the surface membrane, SCCS, and α-granule membranes (30%). Heterodimer forms on activation.

CAM, Cell adhesion molecule; GP, glycoprotein; SCCS, surface-connected canalicular system; VWF, von Willebrand factor.

Several integrins bind collagen, enabling the platelet to adhere to the injured blood vessel lining. Integrins are heterodimeric (composed of two dissimilar proteins) CAMs that integrate their ligands, which they bind on the outside of the cell, with the internal cytoskeleton, triggering activation. GP Ia/IIa, or, using integrin terminology, α2β1, is an integrin that binds the subendothelial collagen that becomes uncovered in the damaged blood vessel wall, promoting adhesion of the platelet to the vessel wall (Figures 13-11 and 13-12). Likewise, α5β1 and α6β1 bind the adhesive endothelial cell proteins laminin andfibronectin, which further promotes platelet adhesion. Another collagen-binding receptor is GP VI, a member of the immunoglobulin gene family, so named because the genes of its members have multiple immunoglobulin-like domains. The unclassified platelet receptor GP IV is a key collagen receptor that also binds the adhesive protein thrombospondin.33

Another adhesion receptor is GP Ib/IX/V, a leucine-rich-repeat family CAM, named for its members’ multiple leucine-rich domains. GP Ib/IX/V arises from the genes GP1BAGP1BBGP5, and GP9. It is composed of two molecules each of GP Ibα, GP Ibβ, and GP IX, and one molecule of GP V. These total seven noncovalently bound subunits. The two copies of subunit GP Ibα bind VWF and support platelettethering(deceleration), necessary in capillaries and arterioles where blood flow shear rates exceed 1000 s–1. The accompanying GP Ibβ molecules cross the platelet membrane and interact with actin-binding protein to provide “outside-in” signaling. Two molecules of GP IX and one of GP V help assemble the four GP Ib molecules. Mutations in GP Ibα, GP Ibβ, or GP IX (but not GP V) are associated with a moderate-to-severe mucocutaneous bleeding disorder, Bernard-Soulier syndrome (Chapter 41). Additionally, VWF deficiency is the basis for the most common inherited bleeding disorder, von Willebrand disease (VWD). VW D also is associated with mucocutaneous bleeding, although the disorder is technically a plasma protein (VWF) deficiency, not a platelet abnormality.34

The subunits of the integrin GP IIb/IIIa (αIIbβ3), are separate and inactive (αIIb and β3) as they are distributed across the plasma membrane, the SCCS, and the internal layer of α-granule membranes. These form their active heterodimer, αIIbβ3, only when they encounter an “inside-out” signaling mechanism triggered by collagen binding to GP VI. Although various agonists may activate the platelet, αIIbβ3 is a physiologic requisite because it binds fibrinogen, generating interplatelet cohesion, called platelet aggregation. Mutations in αIIb or β3 cause a severe inherited mucocutaneous bleeding disorder, Glanzmann thrombasthenia (Chapter 41). The αIIbβ3 integrin also binds VWF, vitronectin, and fibronectin, all adhesive proteins that share the target arginine-glycine-aspartate (RGD) amino acid sequence with fibrinogen.3536

The seven-transmembrane repeat receptors

Thrombin, thrombin receptor activation peptide (TRAP), adenosine diphosphate (ADP), epinephrine, and the eicosanoid synthesis pathway (also called the prostaglandin or the cyclooxygenase pathway) product thromboxane A2 (TXA2) all function individually or together to activate platelets (Figures 13-11 and 13-12). These platelet “agonists” are ligands for the seven-transmembrane repeat receptors (STRs), so named for their unique membrane-anchoring structure. The STRs have seven hydrophobic anchoring domains supporting an external binding site and an internal terminus that interacts with G proteins for outside-in platelet signaling. The STRs are listed in Table 13-5.37

TABLE 13-5

Platelet STR Receptor-Ligand Interaction Coupled to Signaling



G Proteins



Coupled to G1 protein that reduces cAMP; coupled to Gq and G12 proteins that increase IP3 and DAG



Coupled to Gq and G12 proteins that increase IP3 and DAG



Coupled to Gq protein that increases IP3 and DAG



Coupled to G1 protein that reduces cAMP

TPα and TPβ


Coupled to Gq protein that increases IP3 and DAG



Coupled to G1 protein that reduces cAMP; potentiates effects of ADP, thrombin, and TXA2



Coupled to GS protein that increases cAMP to inhibit activation

STRs are named for their peculiar sevenfold membrane anchorage. These receptors mediate “outside-in” platelet activation by transmitting signals initiated by external ligand binding to internal G proteins.ADP, Adenosine diphosphate; cAMP, cyclic adenosine monophosphate;DAG,diacylglycerol; IP3, inositol triphosphate; PAR, protease-activated receptor; PGI2, prostaglandin I2 (prostacyclin); STR, seven-transmembrane receptor; TXA2, thromboxane A2; P2Y1 and P2Y12, ADP receptors; TPα and TPβ, thromboxane receptors; IP, PGI2 receptor.

Thrombin cleaves two STRs, protease-activated receptor 1 (PAR1) and PAR4, that together have a total of 1800 membrane copies on an average platelet. Thrombin cleavage of either of these two receptors activates the platelet through G-proteins that in turn activate at least two internal physiologic pathways, described subsequently. Thrombin also interacts with platelets by binding or digesting two CAMs in the leucine-rich repeat family, GP Ibα and GP V, both of which are parts of the GP Ib/IX/V VWF adhesion receptor.37

There are about 600 copies of the high-affinity ADP receptors P2Y1 and P2Y12 per platelet. These STRs also activate the platelet through the G-protein signaling pathways.38

TPα and TPβ bind TXA2. This interaction produces more TXA2 from the platelet, a G-protein based autocrine (self-perpetuating) system that activates neighboring platelets. Epinephrine binds α2-adrenergic sites that also couple to G-proteins and open up membrane calcium channels. The α2-adrenergic sites function similarly to those located on heart muscle. Finally, the receptor site IP binds prostacyclin(prostaglandin I2, PGI2), a prostaglandin produced from endothelial cells. Prostacyclin enters the platelet and raises the internal cyclic adenosine monophosphate (cAMP) concentration of the platelet, thus blocking platelet activation. The platelet membrane also presents STRs for serotonin, platelet-activating factor, prostaglandin E2, PF4, and β-thromboglobulin.39

Additional platelet membrane receptors

About 15 clinically relevant receptors were discussed in the preceding paragraphs. The platelet supports many additional receptors. The CAM immunoglobulin family includes the ICAMs (CD50, CD54, CD102), which play a role in inflammation and the immune reaction; PECAM (CD31), which mediates platelet–to–white blood cell and platelet–to–endothelial cell adhesion; and FcγIIA (CD32), a low-affinity receptor for the immunoglobulin Fc portion that plays a role in a dangerous condition called heparin-induced thrombocytopenia (Chapter 39).40 P-selectin (CD62) is an integrin that facilitates platelet binding to endothelial cells, leukocytes, and one another.41 P-selectin is found on the α-granule membranes of the resting platelet but migrates via the SCCS to the surface of activated platelets. P-selectin or CD62 quantification by flow cytometry is a successful clinical means for measuring in vivo platelet activation.

Platelet cytoskeleton: Microfilaments and microtubules

A thick circumferential bundle of microtubules maintains the platelet’s discoid shape. The circumferential microtubules (Figures 13-11 and 13-12) parallel the plane of the outer surface of the platelet and reside just within, although not touching, the plasma membrane. There are 8 to 20 tubules composed of multiple subunits of tubulin that disassemble at refrigerator temperature or when treated with colchicine. When microtubules disassemble in the cold, platelets become round, but upon warming to 37° C, they recover their original disc shape. On cross section, microtubules are cylindrical, with a diameter of 25 nm. The circumferential microtubules could be a single spiral tubule.42 Besides maintaining the platelet shape, microtubules move inward on activation to enable the expression of α-granule contents. They also reassemble in long parallel bundles during platelet shape change to provide rigidity to pseudopods.

In the narrow area between the microtubules and the membrane lies a thick meshwork of microfilaments composed of actin (Figures 13-11 and 13-12). Actin is contractile in platelets (as in muscle) and anchors the plasma membrane glycoproteins and proteoglycans. Actin also is present throughout the platelet cytoplasm, constituting 20% to 30% of platelet protein. In the resting platelet, actin is globular and amorphous, but as the cytoplasmic calcium concentration rises, actin becomes filamentous and contractile.

The cytoplasm also contains intermediate filaments, ropelike polymers 8 to 12 nm in diameter, of desmin and vimentin. The intermediate filaments connect with actin and the tubules, maintaining the platelet shape. Microtubules, actin microfilaments, and intermediate microfilaments control platelet shape change, extension of pseudopods, and secretion of granule contents.

Platelet granules: Α-granules, dense granules, and lysosomes

There are 50 to 80 α-granules in each platelet. Unlike the nearly opaque dense granules, α-granules stain medium gray in osmium-dye transmission electron microscopy preparations (Figures 13-11 and 13-12). The α-granules are filled with proteins, some endocytosed, some synthesized within the megakaryocyte and stored in platelets (Table 13-6). Several α-granule proteins are membrane bound. As the platelet becomes activated, α-granule membranes fuse with the SCCS. Their contents flow to the nearby microenvironment, where they participate in platelet adhesion and aggregation and support plasma coagulation.43

TABLE 13-6

Representative Platelet α-Granule Proteins


Coagulation Proteins

Noncoagulation Proteins

Proteins Present in Platelet Cytoplasm and α-Granules








Factor V





Proteins Present in α-Granules but Not Cytoplasm

















Protein C inhibitor


Platelet Membrane-Bound Proteins

Restricted to α-granule membrane





In α-granule and plasma membrane









EGF, Endothelial growth factor; GMP, guanidine monophosphate; GP, glycoprotein; HMWK, high-molecular-weight kininogen; Ig, immunoglobulin; PAI-1, plasminogen activator inhibitor-1; PDCI, platelet-derived collagenase inhibitor; PDGF, platelet-derived growth factor; PECAM-1,platelet–endothelial cell adhesion molecule-1; PF4, platelet factor 4; TGF-β, transforming growth factor-β; VEGF/VPF, vascular endothelial growth factor/vascular permeability factor; VWF, von Willebrand factor; cap1, adenyl cyclase–associated protein.

There are two to seven dense granules per platelet. Also called dense bodies, these granules appear later than α-granules in megakaryocyte differentiation and stain black (opaque) when treated with osmium in transmission electron microscopy (Figures 13-11 and 13-12). Small molecules are probably endocytosed and are stored in the dense granules; these are listed in Table 13-7. In contrast to the α-granules, which employ the SCCS, dense granules migrate to the plasma membrane and release their contents directly into the plasma upon platelet activation. Membranes of dense granules support the same integral proteins as the α-granules—P-selectin, αIIbβ3, and GP Ib/IX/V, for instance—which implies a common source for the membranes of both types of granules.44

TABLE 13-7

Dense Granule (Dense Body) Contents

Small Molecule



Nonmetabolic, supports neighboring platelet aggregation by binding to ADP receptors P2Y1, P2Y12


Function unknown, but ATP release is detectable upon platelet activation


Vasoconstrictor that binds endothelial cells and platelet membranes

Ca2+ and Mg2+

Divalent cations support platelet activation and coagulation

ADP, Adenosine diphosphate; ATP, adenosine triphosphate; P2Y1 and P2Y12, members of the purigenic receptor family (receptors that bind purines).

Platelets also have a few lysosomes, similar to those in neutrophils, 300-nm-diameter granules that stain positive for arylsulfatase, β-glucuronidase, acid phosphatase, and catalase. The lysosomes probably digest vessel wall matrix components during in vivo aggregation and may also digest autophagic debris.

Platelet activation

Although the following discussion seems to imply a linear and stepwise process, adhesion, aggregation, and secretion are often simultaneous.4546

Adhesion: Platelets bind elements of the vascular matrix

As blood flows, vessel walls create stress, or shear force, measured in units labeled s−1. Shear forces range from 500 s−1 in venules and veins to 5000 s−1 in arterioles and capillaries and up to 40,000 s−1 in stenosed (hardened) arteries (Figure 13-13). In vessels where the shear rate is over 1000 s−1, platelet adhesion and aggregation require a defined sequence of events that involves collagen, tissue factor, phospholipid, VWF, and a number of platelet CAMs, ligands, and activators (Figure 13-14).47


FIGURE 13-13 Normal blood flow in intact vessels. RBCs and platelets flow near the center, and WBCs marginate and roll. Endothelial cells and the ECM provide several properties that suppress hemostasis. EC, Endothelial cell; ECM, extracellular matrix; FB, fibroblast; PLT, platelet; RBC, red blood cell; SMC, smooth muscle cell; WBC, white blood cell.


FIGURE 13-14 Initial platelet activation leading to platelet adhesion. The glycoprotein (GP) 1bα portion of the GP Ib/IX/V von Willebrand factor (VWF) receptor site binds VWF (1) and GP VI binds collagen (2). The bound GP VI initiates the release of thromboxane A2 (TXA2) and adenosine diphosphate (ADP, 3), which activate α2β1, an additional collagen receptor (4), stabilizing platelet adhesion, and αIIbβ3, the arginine-glycine-aspartate (RGD) receptor site (5) that binds fibrinogen and VWF to support platelet aggregation.

Injury to the blood vessel wall disrupts the collagen of the extracellular matrix (ECM).48 Damaged endothelial cells release VWF from cytoplasmic storage organelles (Figure 13-15).49 VWF, whose molecular weight ranges from 800,000 to 2,000,000 Daltons “unrolls” like a carpet and adheres to the injured site. Though VWF circulates as a globular protein, it become fibrillar as it unrolls and exposes sites that partially bind the GPIbα portion of the platelet membrane GP Ib/IX/V leucine-rich receptor. This is a reversible binding process that “tethers” or decelerates the platelet. Platelet and VWF interactions remain localized by a liver-secreted plasma enzyme, ADAMTS-13, also called VWF-cleaving protease, that digests “unused” VWF.


FIGURE 13-15 Trauma to the blood vessel wall exposes collagen and tissue factor, triggering platelet adhesion and aggregation. EC, Endothelial cell; ECM, extracellular matrix; FB,fibroblast; PL, phospholipid; PLT, platelet; RBC, red blood cell; SMC,smooth muscle cell; TF, tissue factor; VWF, von Willebrand factor; WBC, white blood cell.

At high shear rates, the VWF-GP Ibα tethering reaction is temporary, and the platelet rolls along the surface unless GP VI comes in contact with the exposed ECM collagen.50 When type I fibrillar collagen binds platelet GP VI, the receptor, which is anchored in the membrane by an Fc receptor–like molecule, triggers internal platelet activation pathways, releasing TXA2 and ADP, an “outside-in” reaction.51 These agonists attach to their respective receptors: TPα and TPβ for TXA2, and P2Y1 and P2Y12 for ADP, triggering an “inside-out” reaction that raises the affinity of integrin α2β1 for collagen. The combined effect of GP Ib/IX/V, GP VI, and α2β1 causes the platelet to become firmly affixed to the damaged surface, where it subsequently loses its discoid shape and spreads.52

The internal platelet activators TXA2 and ADP are also secreted from the platelet to the microenvironment, where they activate neighboring platelets through their respective receptors. Further, they provide inside-out activation of integrin αIIbβ3, the key receptor site for fibrinogen, which assists in platelet aggregation.

Aggregation: Platelets irreversibly cohere

In addition to collagen exposure and VWF secretion, blood vessel injury releases constitutive (integral) tissue factor from endothelial cells. Tissue factor triggers the production of thrombin, which reacts with platelet STRs PAR1 and PAR4. This further activation generates the “collagen and thrombin activated” or COAT platelet, integral to the cell-based coagulation model described in Chapter 37 (Figure 13-16). Meanwhile, integrin αIIbβ3 assembles from its resting membrane units αIIb and β3, binding RGD sequences of fibrinogen and VWF and supports platelet-to-platelet aggregation. P-selectin from the α-granule membranes moves to the surface membrane to further promote aggregation. Platelets lose their shape and extend pseudopods. Membrane phospholipids redeploy with the more polar molecules, especially phosphatidylserine, flipping to the outer layer, establishing a surface for the assembly of coagulation factor complexes. As platelet aggregation continues, membrane integrity is lost, and a syncytium of platelet cytoplasm forms as the platelets exhaust internal energy sources.


FIGURE 13-16 Further activation yields the “collagen and thrombin activated (COAT)” platelet (PLT) leading to aggregation. Platelets become activated by agonists—for example, adenosine diphosphate (ADP), thromboxane A2 (TXA2), collagen (Col), or thrombin (Thr). P-selectin (CD 62) moves from the α-granules to the platelet membrane to support adhesion. The inactive αIIb and β3 units assemble to form the active arginine-glycine-aspartate (RGD) receptor αIIbβ3, which binds fibrinogen (Fg) and von Willebrand factor (VWF).

Platelet aggregation is a key part of primary hemostasis, which in arteries may end with the formation of a “white clot,” a clot composed primarily of platelets and VWF (). Although aggregation is a normal part of vessel repair, white clots often imply inappropriate platelet activation in seemingly uninjured arterioles and arteries and are the pathological basis for arterial thrombotic events, such as acute myocardial infarction, peripheral artery disease, and strokes. The risk of these cardiovascular events rises in proportion to the numbers and avidity of platelet membrane αFigure 13-172β1 and GP VI.53


FIGURE 13-17 In arteries and arterioles, the “white clot” consists of platelets and von Willebrand factor. Though primarily a protective mechanism, the white clot may occlude the vessel, causing acute myocardial infarction, stroke, or peripheral artery disease. EC, Endothelial cell; ECM, extracellular matrix; SMC, smooth muscle cell; FB, fibroblast; PLT, platelet; RBC, red blood cell; WBC, white blood cell; VWF, von Willebrand factor; lines indicate collagen.

The combination of polar phospholipid exposure on activated platelets, platelet fragmentation with cellular microparticle dispersion, and secretion of the platelet’s α-granule and dense granule contents triggers secondary hemostasis, called coagulation (see Chapter 37). Fibrin and red blood cells deposit around and within the platelet syncytium, forming a bulky “red clot” (Figure 13-18). The red clot is essential to wound repair, but it may also be characteristic of inappropriate coagulation in venules and veins, resulting in deep vein thrombosis and pulmonary emboli.


FIGURE 13-18 In veins and venules, the bulky “red clot” consists of platelets, von Willebrand factor, fibrin, and RBCs. Though a protective mechanism, the red clot may occlude the vessel, causing venous thromboembolic disease. EC, Endothelial cell;ECM, extracellular matrix; SMC, smooth muscle cell; FB, fibroblast; PLT, platelet; RBC, red blood cell; WBC, white blood cell; VWF, von Willebrand factor; lines indicate collagen.

Secretion: Activated platelets release granular contents

Outside-in platelet activation through ligand (agonist) binding to integrins, STRs (such as ADP binding to P2Y12), and the immunoglobulin gene product GP VI triggers actin microfilament contraction. Intermediate filaments also contract, moving the circumferential microtubules inward and compressing the granules. Contents of α-granules and lysosomes flow through the SCCS, while dense granules migrate to the plasma membrane, where their contents are secreted (Figure 13-11). The dense granule contents are vasoconstrictors and platelet agonists that amplify primary hemostasis; most of the α-granule contents are coagulation proteins that participate in secondary hemostasis (Tables 13-6 and 13-7).

By presenting polar phospholipids on their membrane surfaces, platelets provide a localized cellular milieu that supports coagulation. Phosphatidylserine is the polar phospholipid on which two coagulation pathway complexes assemble: factor IX/VIII (tenase) and factor X/V (prothrombinase), both supported by ionic calcium secreted by the dense granules. The α-granule contents fibrinogen, factors V and VIII, and VWF (which binds and stabilizes factor VIII) are secreted and increase the localized concentrations of these essential coagulation proteins. Their presence further supports the action of tenase and prothrombinase. Platelet secretions provide for cell-based, controlled, localized coagulation. Table 13-8 lists some additional α-granule secretion products that, although not proteins of the coagulation pathway, indirectly support hemostasis. The lists in Tables 13-6, 13-7, and 13-8 are not exhaustive because more and more platelet granule contents continue to be identified through platelet research activities.

TABLE 13-8

Selected α-Granule Proteins and Their Properties

α-Granule Protein


Platelet-derived growth factor

Supports mitosis of vascular fibroblasts and smooth muscle cells

Endothelial growth factor

Supports mitosis of vascular fibroblasts and smooth muscle cells

Transforming growth factor-β

Supports mitosis of vascular fibroblasts and smooth muscle cells


Adhesion molecule


Adhesion molecule

Platelet factor 4

Heparin neutralization, suppresses megakaryocytopoiesis


Found nowhere but platelet α-granules


Fibrinolysis promotion

Plasminogen activator inhibitor-1

Fibrinolysis control


Fibrinolysis control

Protein C inhibitor

Coagulation control

Platelet activation pathways

G proteins

G proteins control cellular activation for all cells (not just platelets) at the inner membrane surface (). G proteins are αβγ heterotrimers (proteins composed of three dissimilar peptides) that bind guanosine diphosphate (GDP) when inactive. Membrane receptor-ligand (agonist) binding promotes GDP release and its replacement with guanosine triphosphate (GTP). The Gα portion of the three-part G molecule briefly disassociates, exerts enzymatic guanosine triphosphatase activity, and hydrolyzes the bound GTP to GDP, releasing a phosphate radical. The G protein resumes its resting state, but the hydrolysis step provides the necessary phosphorylation trigger to energize the eicosanoid synthesis or the IPFigure 13-193-DAG pathway (Table 13-9).


FIGURE 13-19 G-protein mechanism. (1) A ligand (agonist) binds its corresponding receptor; (2) G-protein swaps GDP for GTP; (3) GTP donates a high energy phosphate radical to a zymogen, remains attached to the G-protein as GDP; (4) the zymogen is activated. The G-protein returns to a resting state bound to GDP. GDP, Guanosine diphosphate.

TABLE 13-9

G Proteins in Platelets and Their Functions

G Protein

Coupled to Receptor

Agonist (Ligand)






Decelerates adenylate cyclase

Reduce cAMP concentration











Activates phospholipase C

Increase IP3-DAG








TPα and TPβ






Activates protein kinase C

Activate pleckstrin, actin microfilaments








TPα and TPβ






Accelerates adenylate cyclase

Increase cAMP concentration

ADP, Adenosine diphosphate; cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; IP3, inositol triphosphate; PAR, protease-activated receptor; TXA2, thromboxane A2P2Y1 and P2Y12, ADP receptors; TPα and TPβ, thromboxane receptors; IP, PGI2 receptor.

Eicosanoid synthesis

The eicosanoid synthesis pathway, alternatively called the prostaglandin, cyclooxygenase, or thromboxane pathway, is one of two essential platelet activation pathways triggered by G proteins (Figure 13-20). The platelet membrane’s inner leaflet is rich in phosphatidylinositol, a phospholipid whose number 2 carbon binds numerous types of unsaturated fatty acids, but especially 5,8,11,14-eicosatetraenoic acid, commonly called arachidonic acid. Membrane receptor-ligand binding and the consequent G-protein activation triggers phospholipase A2, a membrane enzyme that cleaves the ester bond connecting the number 2 carbon of the triglyceride backbone with arachidonic acid. Cleavage releases arachidonic acid to the cytoplasm, where it becomes the substrate for cyclooxygenase, anchored in the DTS. Cyclooxygenase converts arachidonic acid to prostaglandin G2 and prostaglandin H2, and then thromboxane synthetase acts on prostaglandin H2 to produce TXA2. TXA2 binds membrane receptors TPα or TPβ, decelerating adenylate cyclase activity and reducing cAMP concentrations, which mobilizes ionic calcium from the DTS (Figure 13-21). The rising cytoplasmic calcium level causes contraction of actin microfilaments and platelet activation.


FIGURE 13-20 Eicosanoid synthesis. Ligands (agonists) ADP, thrombin, collagen, or epinephrine bind their respective membrane receptors. The combination activates phospholipase A2through the G-protein mechanism described in Figure 13-19. Phospholipase A2 releases arachidonic acid from membrane phosphatidyl inositol. Arachidonic acid is acted upon by cyclooxygenase, peroxidase, and thromboxane synthase to produce TXA2, which activates the platelet through adenylate cyclase, as shown in Figure 13-21. When reagent arachidonic acid is used as an agonist, it bypasses the membrane and directly enters the eicosanoid synthesis pathway. In the endothelial cell, the eicosanoid pathway is nearly identical, except that prostacyclin synthase replaces thromboxane synthase. ADP, Adenosine diphosphate; PgG2, prostaglandin G2PgH2, prostaglandin H2TXA2, thromboxane A2.


FIGURE 13-21 The second messenger system. In the platelet, TXA2 suppresses adenylate cyclase and reduces cyclic AMP concentration. This allows the release of ionic calcium from the DTS. Ionic calcium supports the contraction of actin microfilaments, activating the platelet. In the endothelial cell, prostacyclin (not pictured) has the opposite effect upon adenylate cyclase, raising cyclic AMP and sequestering ionic calcium in the DTS. ATP, Adenosine triphosphate; AMP, adenosine monophosphate; DTS, dense tubular system; TXA2, thromboxane A2.

The cyclooxygenase pathway in endothelial cells incorporates the enzyme prostacyclin synthetase in place of the thromboxane synthetase in platelets. The eicosanoid pathway end point for the endothelial cell is prostaglandin I2, or prostacyclin, which infiltrates the platelet and binds its IP receptor site. Prostacyclin binding accelerates adenylate cyclase, increasing cAMP, and sequesters ionic calcium to the DTS. The endothelial cell pathway suppresses platelet activation in the intact blood vessel through this mechanism, creating a dynamic equilibrium.

TXA2 has a half-life of 30 seconds, diffuses from the platelet, and becomes spontaneously reduced to thromboxane B2, a stable, measurable plasma metabolite. Efforts to produce a clinical assay for plasma thromboxane B2 have been unsuccessful, because special specimen management is required to prevent ex vivo platelet activation with unregulated release of thromboxane B2 subsequent to collection. Thromboxane B2 is acted on by a variety of liver enzymes to produce an array of soluble urine metabolites, including 11-dehydrothromboxane B2, which is stable and measurable.5455

Inositol triphosphate–diacylglycerol activation pathway

The IP3-DAG pathway is the second G protein–dependent platelet activation pathway (Figure 13-22). G-protein activation triggers the enzyme phospholipase C. Phospholipase C cleaves membrane phosphatidylinositol 4,5-bisphosphate to form IP3 and DAG, both second messengers for intracellular activation. IP3 promotes release of ionic calcium from the DTS, which triggers actin microfilament contraction. IP3 may also activate phospholipase A2. DAG triggers a multistep process: activation of phosphokinase C, which triggers phosphorylation of the protein pleckstrin, which regulates actin microfilament contraction.


FIGURE 13-22 PLC is activated by the G-protein mechanism and cleaves the phosphodiester bond from carbon 3 of phosphatidylinositol. One product is DAG, which directly generates actin microfilament contraction. The other product is IP3, which releases ionic calcium from the DTS. DAG, Diacylglycerol; DTS, dense tubular system; IP3, inositol triphosphate; PLC,phospholipase C; R1, saturated fatty acid remains attached to carbon 1; R2, unsaturated fatty acid remains attached to carbon 2. Source: (Courtesy of Larry D. Brace, PhD, Edward Hospital, Naperville, IL.)

Internal platelet activation pathways, like internal pathways of all metabolically active cells, are often called second messengers because they are triggered by a primary ligand-receptor binding event. Second messengers include G proteins, the eicosanoid synthesis pathway, the IP3-DAG pathway, adenylate cyclase, cAMP, and intracellular ionic calcium. This discussion has been limited to activation pathways whose aberrations cause hemostatic disease. The reader is referred to cell physiology texts for a comprehensive discussion of cellular activation pathways.


• Platelets arise from bone marrow megakaryocytes, which reside adjacent to the venous sinusoid. Megakaryocyte progenitors are recruited by IL-3, IL-6, IL-11, and TPO and mature via endomitosis.

• Platelets are released into the bone marrow through shedding from megakaryocyte proplatelet processes, a process called thrombocytopoiesis.

• Circulating platelets are complex anucleate cells with a thick surface glycocalyx bearing an assortment of coagulation factors and plasma proteins.

• Platelets’ SCCS and closely aligned DTS facilitate the storage and release of hemostatic proteins, Ca+2, and enzymes.

• In platelet cytoplasm resides a system of cytoplasmic microfibrils and microtubules that accomplish platelet contraction and pseudopod extension.

• Also within platelet cytoplasm are platelet α-granules and dense granules that store and secrete coagulation factors and vasoactive molecules.

• The platelet membrane supports an array of receptor sites that control platelet activation upon binding their respective ligands.

• Platelets adhere to exposed collagen, aggregate with each other, and secrete the substances stored within their granules.

• Platelet activation is managed internally through G proteins, the eicosanoid synthesis pathway, and the IP3-DAG pathway.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. The megakaryocyte progenitor that undergoes endomitosis is:

a. MK-I

b. BFU-Meg

c. CFU-Meg

d. LD-CFU-Meg

2. The growth factor that is produced in the kidney and induces growth and differentiation of committed megakaryocyte progenitors is:

a. IL-3

b. IL-6

c. IL-11

d. TPO

3. What platelet organelle sequesters ionic calcium and binds a series of enzymes of the eicosanoid pathway?

a. G protein

b. Dense granules

c. DTS


4. What platelet membrane receptor binds fibrinogen and supports platelet aggregation?

a. GP Ib/IX/V

b. GP IIb/IIIa

c. GP Ia/IIa

d. P2Y1

5. What platelet membrane phospholipid flips from the inner surface to the plasma surface on activation and serves as the assembly point for coagulation factors?

a. Phosphatidylethanolamine

b. Phosphatidylinositol

c. Phosphatidylcholine

d. Phosphatidylserine

6. What is the name of the eicosanoid metabolite produced from endothelial cells that suppresses platelet activity?

a. TXA2

b. Arachidonic acid

c. Cyclooxygenase

d. Prostacyclin

7. Which of the following molecules is stored in platelet dense granules?

a. Serotonin

b. Fibrinogen

c. PF4

d. Platelet-derived growth factor

8. What plasma protein is essential to platelet adhesion?

a. VWF

b. Factor VIII

c. Fibrinogen

d. P-selectin

9. Reticulated platelets can be enumerated in peripheral blood to detect:

a. Impaired production in disease states

b. Abnormal organelles associated with diseases such as leukemia

c. Increased platelet production in response to need

d. Inadequate rates of membrane cholesterol exchange with the plasma

10. Platelet adhesion refers to platelets:

a. Sticking to other platelets

b. Releasing platelet granule constituents

c. Providing the surface for assembly of coagulation factors

d. Sticking to surfaces such as subendothelial collagen


1.  Butkiewicz A. M, Kemona H, Dymicka-Piekarska V, et al. Platelet count, mean platelet volume and thrombocytopoietic indices in healthy women and menThromb Res; 2006; 118:199-204.

2.  Lance M. D, Sloep M, Henskens Y. M, Marcus M. A. Mean platelet volume as a diagnostic marker for cardiovascular disease drawbacks of preanalytical conditions and measuring techniques. Clin Appl Thromb Hemost; 2012; 18:561-568.

3.  Reddy V, Marques M. B, Fritsma G. A. Quick Guide to Hematology Testing. 2nd Ed. Washington, DC : AACC Press 2013.

4.  Deutsch V. R, Tomer A. Advances in megakaryocytopoiesis and thrombopoiesis from bench to bedside. Br J Haematol; 2013; 161:778-793.

5.  Kosaki G. In vivo platelet production from mature megakaryocytes does platelet release occur via proplatelets. Int J Hematol; 2005; 81:208-219.

6.  Chang Y, Bluteau D, Bebili N. From hematopoietic stem cells to plateletsJ Thrombos Haemostas; 2007; 5(Suppl. 1):318-327.

7.  Italiano J. E, Jr Hartwig J. H. Megakaryocyte structure and platelet biogenesis. In: Marder V. J, Aird W. C, Bennett J. S, Schulman S, White G. C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice.6th ed. Philadelphia : Lippincott Williams & Wilkins 2012; 365-372.

8.  Lordier L, Jalil A, Aurade F. Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signalingBlood; 2008; 112:3164-3174.

9.  Italiano J. E, Hartwig J. H. Megakaryocyte and platelet structure. In: Hoffman R, Benz E. J, Silbestein L. E, Heslop H. E, Weitz J. I, Anastasi J. Hematology: Basic Principles and Practice. 6th ed. St. Louis : Elsevier 2013; 1797-1808.

10.  Ramstrom A. S, Fagerberg I. H, Lindahl T. L. A flow cytometric assay for the study of dense granule storage and release in human plateletsPlatelets; 1999; 10:153-158.

11.  Della Porta M. G, Lanza F, Del Vecchio L, Italian Society of Cytometry (GIC). Flow cytometry immunophenotyping for the evaluation of bone marrow dysplasiaCytometry B Clin Cytom; 2011; 80:201-211.

12.  Berndt M. C, Andrews R. K. Major platelet glycoproteins platelet glycoprotein Ib-IX-V. In: Marder V. J, Aird W. C, Bennett J. S, Schulman S, White G. C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 6th ed. Philadelphia : Lippincott Williams & Wilkins 2012; 382-385.

13.  Yee F, Ginsberg M. H. Major platelet glycoproteins Integrin aIIb b3  (GP IIb-IIIa). In: Marder V.J, Aird W.C, Bennett J.S, Schulman S, White G.C. Hemostasis and Thrombosis Basic Principles and Clinical Practice 6th ed. Philadelphia : Lippincott Williams & Wilkins 2012; 386-392.

14.  Junt T, Schulze H, Chen Z. Dynamic visualization of thrombopoiesis within bone marrowScience; 2007; 317:1767-1770.

15.  Kuter D. J. Biology and chemistry of thrombopoietic agentsSemin Hematol; 2010; 47:243-248.

16.  Kaushansky K. The molecular mechanisms that control thrombopoiesisJ Clin Invest; 2005; 115:3339-3347.

17.  Neunert C, Lim W, Crowther M. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopeniaBlood; 2011; 117:4190-4207.

18.  Bussel J. B, Pinheiro M. P. EltrombopagCancer Treatment and Research; 2011; 157:289-303.

19.  Sitaraman S. V, Gewirtz A. T. Oprelvekin. Genetics InstituteCurr Opin Investig Drugs; 2001; 2:1395-1400.

20.  Stasi R. Therapeutic strategies for hepatitis- and other infection-related immune thrombocytopeniasSemin Hematol; 2009; 46(Suppl. 2):S15-S25.

21.  Yu M, Cantor A. B. Megakaryopoiesis and thrombopoiesis an update on cytokines and lineage surface markers. Methods Mol Biol; 2012; 788:291-303.

22.  Leader, A, Pereg, D, & Lishner, M. Are platelet volume indices of clinical use? A multidisciplinary review. Ann Med, 44, 805–816.

23.  Rodak B. F, Carr J. H. Clinical Hematology Atlas. 4th ed. St. Louis : Elsevier 2013.

24.  Michur H, Mas´lanka K, Szczepin´ski A, Marian´ska B. Reticulated platelets as a marker of platelet recovery after allogeneic stem cell transplantationInt J Lab Hematol; 2008; 30:519-525.

25.  Tsiara S, Elisaf M, Jagroop I. A. Platelets as predictors of vascular risk is there a practical index of platelet activity. Clin Appl Thromb Hemost; 2003; 9:177-190.

26.  Briggs C, Harrison P, Machin S. J. Continuing developments with the automated platelet countInt J Lab Hematol; 2007; 29:77-91.

27.  Abe Y, Wada H, Sakakura M, et al. Usefulness of fully automated measurement of reticulated platelets using whole bloodClin Appl Thromb Hemost; 2005; 11:263-270.

28.  Briggs C, Kunka S, Hart D, et al. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopeniaBr J Haematol; 2004; 126:93-99.

29.  Cesari F, Marcucci R, Gori A. M, et al. Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Insights from the AMI-Florence 2 StudyThromb Haemost; 2013; 109:846-853.

30.  Kunicki T. J, Nugent D. J. Platelet glycoprotein polymorphisms and relationship to function, immunogenicity, and disease. In: Marder V. J, Aird W. C, Bennett J. S, Schulman S, White G. C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 6th ed. Philadelphia : Lippincott Williams & Wilkins 2012; 393-399.

31.  Zieseniss S, Zahler S, Muller I, et al. Modified phosphatidylethanolamine as the active component of oxidized low density lipoprotein promoting platelet prothrombinase activityJ Biol Chem; 2001; 276:19828-19835.

32.  Savage B, Ruggeri Z. V. The basis for platelet adhesion. In: Marder V. J, Aird W. C, Bennett J. S, Schulman S, White G. C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 6th ed. Philadelphia : Lippincott Williams & Wilkins 2012; 400-449.

33.  Tandon N. N, Kraslisz U, Jamieson G. A. Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesionJ Biol Chem; 1989; 264:7576-7583.

34.  Rivera J, Lozano M. L, Corral J, et al. Platelet GP Ib/IX/V complex physiological role. J Physiol Biochem; 2000; 56:355-365.

35.  Jobe S, Di Paola J. Congenital and acquired disorders of platelet function and number. In: Kitchens C. S, Kessler C. M, Konkle B. A. Consultative Hemostasis and Thrombosis. 3rd ed. St. Louis : Elsevier 2013; 132-149.

36.  Granada J. F, Kleiman N. S. Therapeutic use of intravenous eptifibatide in patients undergoing percutaneous coronary intervention acute coronary syndromes and elective stenting. Am J Cardiovasc Drugs; 2004; 4:31-41.

37.  Jackson, S. P, Nesbitt, W. S, & Kulkarni, S. Signaling events underlying thrombus formation. J Thromb Haemost, 1, 1602–1612.

38.  Moliterno D. J. Advances in antiplatelet therapy for ACS and PCIJ Interven Cardiol; 2008; 21(Suppl. 1):S18-S24.

39.  Israels S. J, Rand M. L. What we have learned from inherited platelet disordersPediatr Blood Cancer; 2013; 60(Suppl. 1):S2-S7.

40.  Walenga J. M, Jeske W. P, Prechel M. M, et al. Newer insights on the mechanism of heparin-induced thrombocytopeniaSemin Thromb Hemost; 2004; 30(Suppl. 1):57-67.

41.  Keating F. K, Dauerman H. L, Whitaker D. A, et al. Increased expression of platelet P-selectin and formation of platelet-leukocyte aggregates in blood from patients treated with unfractionated heparin plus eptifibatide compared with bivalirudinThromb Res; 2006; 118:361-369.

42.  White J. G, Rao G. H. Microtubule coils versus the surface membrane cytoskeleton in maintenance and restoration of platelet discoid shapeAm J Pathol; 1998; 152:597-609.

43.  Blair P, Flaumenhaft R. Platelet alpha-granules basic biology and clinical correlates. Blood Rev; 2009; 23:177-189.

44.  Abrams C. S, Plow E. F. Molecular basis for platelet function. In: Hoffman R. H, Benz E. J, Silberstein L. E. Hematology: Basic Principles and Practice. 6th ed. St. Louis : Elsevier 2013; 1809-1820.

45.  Ye S, Whiteheart S. W. Molecular basis for platelet secretion. In: Marder V. J, Aird W. C, Bennett J. S, Schulman S, White G. C. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 6th ed. Philadelphia : Lippincott Williams & Wilkins 2012; 441-449.

46.  Abrams C. S. Intracellular signaling in plateletsCurr Opin Hematol; 2005; 12:401-405.

47.  Stegner D, Nieswantdt B. Platelet receptor signaling in thrombus formationJ Mol Med; 2011; 89:109-121.

48.  Tailor A, Cooper D, Granger D. N. Platelet-vessel wall interactions in the microcirculationMicrocirculation; 2005; 12:275-285.

49.  Zhou Z, Nguyen T. C, Guchhait P, Dong J. F. Von Willebrand factor, ADAMTS-13, and thrombotic thrombocytopenic purpuraSemin Thromb Hemost; 2010; 36:71-81.

50.  Nieswandt B, Watson S. P. Platelet-collagen interaction is GPVI the central receptor. Blood; 2003; 102:449-461.

51.  Varga-Szabo D, Pleines I, Nieswandt B. Cell adhesion mechanisms in plateletsArterioscler Thromb Vasc Biol; 2008; 28:403-412.

52.  Jung S. M, Moroi M, Soejima K, et al. Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing bloodJ Biol Chemistry; 2012; 287:30000-30013.

53.  Furihata K, Nugent D. J, Kunicki T. J. Influence of platelet collagen receptor polymorphisms on risk for arterial thrombosisArch Pathol Lab Med; 2002; 126:305-309.

54.  Fritsma G. A, Ens G. E, Alvord M. A, et al. Monitoring the antiplatelet action of aspirinJAAPA; 2001; 14:57-62.

55.  Eikelboom J. W, Hankey G. J. Failure of aspirin to prevent atherothrombosis potential mechanisms and implications for clinical practice. Am J Cardiovasc Drugs; 2004; 4:57-67.