Endomitosis

Megakaryocyte maturation is marked by a mysterious form of mitosis that lacks telophase and cytokinesis (separation into daughter cells) and is called endomitosis. In endomitosis, DNA replication proceeds to the production of 8N, 16N, or 32N ploidy with duplicated sets of chromosomes, but no cell division.⁶ Some megakaryocyte nuclei replicate five times, reaching 128N; this level of ploidy is unusual, however, and may signal hematologic disease. Megakaryocytes employ their copious DNA to synthesize abundant cytoplasm, which differentiates into platelets. A single megakaryocyte may shed 2000 to 4000 platelets. In an average-size healthy human there are 10⁸ megakaryocytes producing 10¹¹ platelets per day. The key to endomitosis is the loss of spindle fiber orientation at the point of telophase, so that chromosomes do not proceed from equatorial plates to polar bodies, but rather duplicate in place. Cytokinesis is arrested, a cell-cycle adaptation found in no other normal human cell.

Megakaryocyte Progenitors

The megakaryocyte-erythrocyte progenitor (see Figure 7-13) differentiates into the megakaryocyte lineage under the influence of the hormone thrombopoietin (TPO) and a series of cytokines (see Table 13-3). There are three megakaryocyte lineage-committed progenitor stages, defined by their culture colony characteristics. In order of differentiation, these are the least mature burst-forming unit (BFU-Meg), the colony-forming unit (CFU-Meg), and the most mature light-density CFU (LD-CFU-Meg).⁷ All three progenitor stages resemble small 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 pool of megakaryocyte progenitors. Their proliferative (mitotic) properties are reflected in their ability to form colonies of hundreds (BFUs) or scores (CFUs) of progeny in culture (Figure 13-2). In contrast to the BFU-Meg and CFU-Meg, the LD-CFU-Meg has little proliferative capacity and produces few cells, but begins the progress through endomitosis to reach increased nuclear ploidy.

The LD-CFU-Meg may be a transitional, or “promegakaryoblast,” stage in which polyploidy is first established, but its morphology is indistinguishable from that of small lymphocytes. At the LD-CFU-Meg point of development, megakaryocyte progenitors enter terminal differentiation, as they lose their ability to undergo normal mitosis but continue with endomitosis.

In specialty laboratories, immunologic probes and flow cytometry are employed to identify megakaryocyte progenitors. Useful flow cytometric progenitor markers are the general stem cell marker CD34, HLA-DR, and platelet glycoprotein IIIa (GP IIb/IIIa, CD41).⁸ Platelet peroxidase, localized in the endoplasmic reticulum of progenitors and megakaryoblasts, also may be identified by cytochemical stain in transmission electron microscopy. Identical peroxidase activity is localized to the dense tubular system of mature platelets.⁹

Terminal Megakaryocyte Differentiation

Megakaryocyte progenitors leave the proliferative phase and enter terminal differentiation, a series of stages in which microscopists begin 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).

Morphologists call the least differentiated megakaryocyte precursor the MK-I stage or megakaryoblast. Although they no longer look like small lymphocytes, megakaryoblasts cannot be reliably distinguished from myeloblasts or pronormoblasts using light microscopy (Figure 13-4). The morphologist may see plasma membrane blebs, blunt projections from the margin that resemble platelets. At this stage the megakaryocyte begins to develop most of its cytoplasmic ultrastructure, including procoagulant-laden α-granules, δ-granules (dense bodies), and the demarcation system (DMS).¹⁰

The later section on mature platelet ultrastructure discusses α-granules and δ-granules with their contents. The DMS is a series of membrane-lined channels that invade from the plasma membrane and grow over the course of terminal differentiation to subdivide the entire cytoplasm. The DMS is biologically identical to the plasma membrane and ultimately delineates the individual platelets during thrombocytopoiesis.

If there is a clinical need, immunologic probes or cytochemical markers are employed for definitive identification by flow cytometry (Table 13-2). In addition to the progenitor markers listed previously, the megakaryoblast may be identified using immunologic probes for the more differentiated membrane structures GP Ib, which is part of the GP Ib/IX/V von Willebrand factor (VWF) adhesion receptor (CD42); mpl, which is the TPO receptor site; or cytoplasmic VWF, detected by histochemical immunostaining rather than flow cytometry. CD36, which is GP IV, becomes visible in the developing MK-II stage, and fibrinogen may be detected by immunostaining in the fully developed megakaryocyte.

Resting Platelet Plasma Membrane

The platelet plasma membrane resembles any biologic membrane: a bilayer composed of proteins and lipids, as diagrammed in Figure 13-9. 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.

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 is converted to the eicosanoids prostaglandin and thromboxane during platelet activation. Phosphatidylserine flips to the outer surface on activation and is the phospholipid surface on which coagulation enzymes assemble.²⁴

Esterified cholesterol moves freely throughout the hydrophobic internal layer, exchanging with unesterified plasma cholesterol. 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 in 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 requirements. The platelet literally 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). The SCCS twists spongelike throughout the platelet, enabling the platelet to store additional quantities of the hemostatic proteins of the glycocalyx, raising its capacity manyfold. The glycocalyx is less developed in the SCCS and lacks some of the glycoprotein receptors present on the surface. The SCCS is the route for endocytosis and for secretion of granular contents upon platelet activation.

Dense Tubular System

Parallel and closely aligned to the SCCS is the dense tubular system (DTS), a condensed remnant of the rough endoplasmic reticulum. 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 prostaglandin synthesis, 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 gene family, the CAM selectin family, the seven-transmembrane receptor (STR) family, and some miscellaneous receptors.²⁵ Table 13-4 lists the receptors that support the initial phases of platelet adhesion and aggregation.

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 α₂β₁, is an integrin that binds subendothelial collagen in the damaged blood vessel wall to promote adhesion of the platelet to the vessel wall in regions of low blood flow, veins whose laminar flow shear rates are less than 1000 s^–1. Likewise, α₅β₁ and α₆β₁ bind the adhesive endothelial cell proteins laminin and fibronectin, 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 also binds collagen and the adhesive protein thrombospondin.²⁶

Perhaps the most important 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 GP1BA, GP1BB, GP9, and GP5. It is composed of two molecules each of GP Ibα, GP Ibβ, and GP IX, and one molecule of GP V. This totals seven noncovalently-bound subunits. The two copies of subunit GP Ibα account for VWF binding, necessary in regions with blood flow shear rates higher than 1000 s^–1, as are found in capillaries and arterioles. Additional sites on the GP Ibα molecule bind thrombin. 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 hold the four GP Ib molecules together. 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. VWF deficiency is the basis for the most common inherited bleeding disorder, von Willebrand disease. Von Willebrand disease also is associated with mucocutaneous bleeding, although it is technically a plasma protein deficiency, not a platelet abnormality.²⁷

The two subunits of GP IIb/IIIa, α_IIb and β₃, are initially inactive as they are distributed across the plasma membrane, the SCCS, and the internal layer of α-granule membranes. These form an active heterodimer, α_IIbβ₃, only when they encounter an “inside-out” signaling mechanism triggered by collagen binding to GP VI or VWF binding to GP Ib/IX/V. Although various agonists may activate the platelet, α_IIbβ₃ is a physiologic requisite because it binds fibrinogen, generating interplatelet aggregation. Mutations in α_IIb or β₃ cause a severe inherited mucocutaneous bleeding disorder, Glanzmann thrombasthenia. The α_IIbβ₃ integrin also binds VWF, vitronectin, and fibronectin, all adhesive proteins that share the target arginine-glycine-aspartate (RGD) amino acid sequence with fibrinogen.²⁸ GP IIb/IIIa (α_IIbβ₃) is the target receptor for the intravenous “glycoprotein inhibitor” (GPI) drugs ReoPro (abciximab, Eli Lilly), Integrilin (eptifibatide, Millennium Pharmaceuticals) and Aggrastat (tirofiban, Medicure Pharma), any one of which may be employed during cardiac catherization. Use of the glycoprotein inhibitors requires platelet function assays for efficacy and safety (see Chapter 46).

Platelet Plasma Membrane Receptors That Provide for Activation: The Seven-Transmembrane Receptors

Thrombin, thrombin receptor activation peptide (TRAP), adenosine diphosphate (ADP), epinephrine, and the prostaglandin (eicosanoid, cyclooxygenase) pathway product thromboxane A₂ (TXA₂) all activate platelets. These platelet “agonists” are ligands for 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 13-5.²⁹

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 at least two internal physiologic pathways, described later in the Platelet Activation section. Thrombin also interacts with platelets by binding or digesting two CAMs in the leucine-rich repeat family, GP Ibα (which is part of the GP Ib/IX/V VWF adhesion site) and GP V.

There are about 600 copies of the high-affinity ADP receptors P2Y₁ and P2Y₁₂. These STRs activate the platelet through the G-protein signaling pathways. Platelets are partially inactivated by prasugrel (Effient), clopidogrel (Plavix) and ticlopidine (Ticlid), drugs that occupy P2Y₁₂ (see Chapter 46). These drugs are standard oral antithrombotic treatments prescribed after acute myocardial infarctions, ischemic cerebrovascular accidents, and venous thromboembolic events. Platelet function assays are regularly employed to monitor prasugrel and clopidogrel efficacy and establish dosages.³⁰

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

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 FcγRIIA (CD32), a low-affinity receptor for the immunoglobulin Fc portion that plays a role in a dangerous condition called heparin-induced thrombocytopenia (see Chapter 42).³¹ P-selectin (CD62) is an integrin that encourages platelets to bind endothelial cells, leukocytes, and each other.³² 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 shape. The circumferential microtubules parallel the plane of the disc 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, 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, and hollow. The circumferential microtubules could be a single spiral tubule.³³ Besides maintaining platelet shape, microtubules contract on activation to encourage expression of α-granule contents. They also reassemble in long parallel bundles to provide rigidity to pseudopods.

In the narrow area between the microtubules and membrane lies a thick meshwork of microfilaments composed of actin. 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 platelet shape. Microtubules, actin microfilaments, and intermediate microfilaments control platelet shape change, extension of pseudopods, and secretion of granule contents.

Platelet Granules: α-Granules, δ-Granules, and Lysosomes

There are 50 to 80 α-granules in each platelet. In contrast to the nearly opaque δ-granules, α-granules stain medium gray in osmium-dye transmission electron microscopy preparations (Figure 13-10). 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 adhesion and aggregation and support plasma coagulation.

Gray platelet syndrome is an inherited absence of α-granule contents. The granule membranes are present with their membrane-bound proteins, but the anticipated soluble proteins normally within are missing. In gray platelet syndrome, platelets appear large and light gray in Wright-stained blood films. Platelet aggregation in response to ADP, collagen, epinephrine, and thrombin is diminished, and patients experience moderate to severe lifelong mucocutaneous bleeding.

There are two to seven δ-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. Small molecules are probably endocytosed and are stored in the δ-granules; these are listed in Table 13-7. In contrast to the α-granules, which employ the SCCS, δ-granules migrate to the plasma membrane and release their contents directly into the plasma upon platelet activation. Membranes of δ-granules support the same integral proteins as the α-granules—P-selectin, GP IIb/IIIa, and GP Ib/IX/V, for instance—which implies a common source for the membranes of both types of granules.

Storage pool disorder is the name given to diminished δ-granule contents. Storage pool disorders occur in inherited diseases characterized by albinism, such as Hermansky-Pudlak and Chédiak-Higashi syndromes, as well as in Wiskott-Aldrich syndrome, a nonalbinism X-linked recessive δ-granule deficiency characterized by severe eczema. The inherited storage pool disorders are associated with mild mucocutaneous bleeding.

More commonly, storage pool disorder arises with irregular platelet production in myeloproliferative neoplasms or myelodysplastic syndromes. In these hematologic diseases, both α-granules and δ-granules are reduced and abnormal in size, shape, and contents. There may be mucocutaneous bleeding or even thrombosis.

Storage pool disorder (δ-granule deficiency) does not affect Wright stain platelet morphology, but platelets fail to secrete when treated with thrombin or TRAP in lumiaggregometry. Mepacrine, a quinidine-derived fluorescent dye, stains δ-granule phosphates, which allows quantitation in flow cytometry.

Refer to Chapter 44 for more information on platelet granules, gray platelet syndrome, and storage pool disorder.

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.

Adhesion: Platelets Reversibly Bind Elements of the Vascular Matrix

Platelets repair minor injuries to blood vessel linings, such as regular sloughing of senescent endothelial cells, through adhesion (Figures 13-11 and 13-12).³⁶ Platelets may adhere directly to collagen of the vascular matrix in veins or venules through the CAMs described previously: GP Ia/IIa, GP IV, and GP VI.

In capillaries and arterioles, where the blood flows rapidly and the shear rate exceeds 1000 s⁻¹, the site of injury is first “carpeted” by VWF. VWF circulates as a multimeric globulin with a molecular weight of between 800,000 D and 20 million D, one of the largest proteins in the plasma. The combination of injury and shear stress “unrolls” the globular molecule to form fibrillar strands that coat injury sites and bind subendothelial collagen. A liver-secreted plasma enzyme, VWF-cleaving protease, controls thrombogenicity by rapidly digesting fibrillar VWF to inactive fragments.³⁷ Platelets adhere to fibrillar VWF through their GP Ib/IX/V receptor, and the VWF-platelet layer fills in the space made by the injury to await replacement by normal vascular cells (Figure 13-13). Although seldom an isolated process, adhesion alone may exclude secretion of granule contents or the energy-dependent redistribution of CAMs such as GP IIb/IIIa (α_IIbβ₃) or P-selectin. Adhesion does require contraction of actin molecules, however, and the formation of pseudopods.

Although history records several attempts, no one has devised a clinical laboratory method that isolates and measures platelet adhesion without simultaneously involving aggregation and secretion. Clinicians routinely employ laboratory instrumentation to measure VWF activity and concentration, however, and clinicians may quantitate several CAMs through flow cytometry in specialized laboratories.

Aggregation: Platelets Irreversibly Bind Each Other

When vessel damage is extensive, platelets adhere and aggregate (Figures 13-14 and 13-15). Aggregation requires the active conformation of the GP IIb/IIIa (α_IIbβ₃) integrin and includes pseudopod formation and redistribution of P-selectin to the surface membrane (Figure 13-16). Membrane phospholipids also redeploy, with the more polar molecules, such as phosphatidylserine, flipping to the outer layer. GP IIb/IIIa binds the RGD sequence of any plasma protein; the protein most readily available is fibrinogen, which is the major player in aggregation. Membrane integrity is lost, and a syncytium of platelet cytoplasm forms as the platelet exhausts internal energy sources. Aggregation is a part of primary hemostasis, and its irreversible end point is the “white clot” or platelet-VWF plug. Although a normal part of vessel repair, white clots often imply inappropriate platelet activation in uninjured arterioles and arteries and are the pathologic basis for arterial thrombotic events, such as acute myocardial infarction, peripheral vascular disease, and strokes. Except for VWF, coagulation proteins are excluded from white clots.

The combination of polar phospholipid exposure, platelet microparticle dispersion, and secretion of the platelet’s α- and δ-granule contents triggers secondary hemostasis, called coagulation (see Chapter 40). Fibrin and red blood cells deposit around and within the platelet syncytium, forming a bulky “red clot.” The red clot is essential to wound repair, but is characteristic of inappropriate coagulation in venules and veins, resulting in deep vein thrombosis and pulmonary emboli.

Specialty and tertiary care coagulation laboratories provide in vitro whole blood or plasma platelet aggregometry and lumiaggregometry as a means for detecting and identifying aggregation abnormalities. The bleeding time test, although still used, has limited predictive value. Several systems for the physician’s office or near-patient laboratory, such as the Siemens PFA-100 (Deerfield, Ill.), and the Chronolog WBA (Havertown, Pa.), also are available to test for platelet function.³⁸

Secretion: Activated Platelets Release Granular Contents

Outside-in platelet activation through ligand (agonist) binding to integrins and STRs triggers actin microfilament contraction. Intermediate filaments and microtubules contract, compressing granules. Contents of α-granules and lysosomes flow through the SCCS; meanwhile δ-granule contents are secreted through the plasma membrane (Figure 13-17). The δ-granule contents are vasoconstrictors and platelet agonists that amplify primary hemostasis; several of the α-granule contents are coagulation proteins (Table 13-8).

By presenting polar phospholipids on their membrane surfaces, platelets provide a localized cellular milieu that supports coagulation. Phosphatidylserine is the phospholipid on which two coagulation pathway complexes form: factor IX/VIII (tenase) and factor X/V (prothrombinase), supported by ionic calcium secreted by the δ-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, which further supports the action of tenase and prothrombinase. Platelet secretions serve cell-based, controlled, localized coagulation. Table 13-8 lists some additional α-granule secretions that, although not proteins of the coagulation pathway, support hemostasis. Some proteins listed in Table 13-8 do not appear in Table 13-5. Neither list is exhaustive, because more α-granule contents are being identified.

There are several clinical laboratory assays of platelet secretions. The most successful is lumiaggregometry, which employs firefly luciferase to identify adenosine triphosphate secreted from δ-granules through luminescence. Immunoassays of PF4 or β-thromboglobulin, unique to platelets and secreted by δ-granules, are available, but only from reference laboratories using specialized blood specimen collection techniques. This is because uncontrolled ex vivo platelet activation factitiously elevates the levels of these substances in specimens that are collected and handled using routine processes.

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 protein (Figure 13-19). The platelet membrane’s inner leaflet is rich in phosphatidylinositol, a phospholipid whose number 2 carbon binds numerous unsaturated fatty acids, including 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, 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 13-20). The rising cytoplasmic calcium level causes contraction of actin microfilaments and platelet activation.

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 binds the platelet IP receptor site. Prostacyclin binding accelerates adenylate cyclase, increasing cAMP, and moves ionic calcium to DTS sequestration. The endothelial cell pathway controls platelet activation in the intact blood vessel through this mechanism.

Deficiencies in calcium mobilization, cyclooxygenase, and thromboxane synthetase activity reduce TXA₂ production and platelet activation, an inherited set of conditions identified collectively as platelet release disorder or aspirin-like disorder, which is associated with mucocutaneous bleeding and intracranial hemorrhages. Aggregometry patterns in these disorders mimic the effect of aspirin, which causes a reduced response to ADP, arachidonic acid, and epinephrine. Aspirin also reduces the response to low-concentration (1 mg/mL) collagen, whereas aspirin-like disorders reduce aggregation response at both low and high (5 mg/mL) concentrations of collagen.

TXA₂ has a half-life of 30 seconds, diffuses from the platelet, and spontaneously reduces 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 factitious unregulated release of thromboxane B₂. 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.³⁹ Immunoassays of urine 11-dehydrothromboxane B₂ are used to characterize in vivo platelet activation and the suppressing effect of aspirin.⁴⁰

Inositol Triphosphate–Diacylglycerol Activation Pathway

The IP₃-DAG pathway is the second G protein–dependent platelet activation pathway (Figure 13-21). 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 also may activate phospholipase A₂. DAG triggers a multistep process: activation of phosphokinase C, which triggers phosphorylation of the protein pleckstrin, which regulates actin microfilament contraction.

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 aberration cause hemostatic disease. The reader is referred to cell physiology texts for a comprehensive discussion of cellular activation pathways.