Platelet Structure and Function

125

VOL 28, NO 2 SPRING 2015 CLINICAL LABORATORY SCIENCE

FOCUS: ANTIPLATELET DRUGS AND PLATELET FUNCTION TESTING

Platelet Structure and Function

GEORGE A. FRITSMA

LEARNING OBJECTIVES

1. Diagram platelet structure, including glycocalyx,

plasma membrane, filaments, microtubules, and

granules.

2. Illustrate platelet adhesion, including the role of

von Willebrand factor

3. Illustrate platelet aggregation, including the role of

fibrinogen

4. List the secretions of platelet dense bodies and α-

granules

5. Demonstrate the relationship of platelets and the

plasma coagulation mechanism.

ABBRVIATIONS: ADP-adenosine diphosphate; ATP-

adenosine triphosphate; CAM-cell adhesion molecule;

cAMP-cyclic adenosine monophosphate; DAG-

diacylglycerol; DTS-dense tubular system; ECM-

extracellular matrix; EGF-endothelial growth factor;

GMP-guanidine monophosphate; GP-glycoprotein;

HMWK-high-molecular-weight kininogen; Ig-

immunoglobulin; IP3-inositol triphosphate; IP-PGI2

receptor; MPV-mean platelet volume; P2Y1 and P2Y12-

ADP receptors; PAI-1-plasminogen activator inhibitor-

1; PAR-protease-activated receptor; PF4-platelet factor

4; PGG2-prostaglandin G2; PGH2-prostaglandin H2;

PDCI-platelet-derived collagenase inhibitor; PDGF-

platelet-derived growth factor; PECAM-1-platelet–

endothelial cell adhesion molecule-1; PGI2-

prostaglandin I2 (prostacyclin); RGD-arginine-glycine-

aspartic acid receptor target; SCCS-surface-connected

canalicular system; STR-seven-transmembrane repeat

receptor; TGF-β-transforming growth factor-β; TPα

and TPβ-thromboxane receptors; TXA2-thromboxane

A2; VEGF/VPF-vascular endothelial growth

factor/vascular permeability factor; VWF-von

Willebrand factor

INDEX TERMS: Cell adhesion molecules, eicosanoid

synthesis, glycoprotein, ligands, prostaglandin, platelet

adhesion, platelet aggregation, platelet agonists, platelet

count, platelet function, platelet production, platelet

secretion, platelet structure

Clin Lab Sci 2015;28(2):125

George A. Fritsma, MS, MLS, The Fritsma Factor, Your

Interactive Hemostasis Resource, Fritsma & Fritsma LLC,

Birmingham, AL

Address for Correspondence: George A. Fritsma, MS,

MLS, The Fritsma Factor, Your Interactive Hemostasis

Resource, Fritsma & Fritsma LLC, 153 Redwood Drive,

Birmingham, AL 35173, George@fritsmafactor.com

Platelets are blood cells that are released from bone

marrow megakaryocytes and circulate for approximately

10 days. They possess granular cytoplasm with no

nucleus and their diameter when seen in a Wright-

stained peripheral blood film averages 2.5 um with a

subpopulation of larger cells, 4–5 um. Mean platelet

volume (MPV), as measured in a buffered isotonic

suspension flowing through the impedance-based

detector cell of a clinical profiling instrument, is 8–10

fL.

Circulating, resting platelets are biconvex, although in

EDTA blood they tend to “round up.” On a blood

film, platelets appear circular to irregular, lavender, and

granular, although their diminutive size makes them

hard to examine for internal structure.1 In the blood,

their surface is even, and they flow smoothly through

veins, arteries, and capillaries.

The normal peripheral blood platelet count is 150–

400,000/µL. This count represents only two thirds of

available platelets because the spleen sequesters the

remainder. In hypersplenism or splenomegaly, increased

sequestration may cause a relative thrombocytopenia.

Under conditions of hemostatic need, platelets move

from the spleen to the peripheral blood and answer

cellular and humoral stimuli by becoming irregular and

sticky, extending pseudopods, and adhering to

neighboring structures or aggregating with one another.

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GEORGE A. FRITSMA

LEARNING OBJECTIVES

Diagram platelet structure, including glycocalyx, plasma membrane, filaments, microtubules, and granules.
Illustrate platelet adhesion, including the role of von Willebrand factor
Illustrate platelet aggregation, including the role of fibrinogen
List the secretions of platelet dense bodies and α- granules
Demonstrate the relationship of platelets and the plasma coagulation mechanism. ABBRVIATIONS: ADP-adenosine diphosphate; ATP- adenosine triphosphate; CAM-cell adhesion molecule; cAMP-cyclic adenosine monophosphate; DAG- diacylglycerol; DTS-dense tubular system; ECM- extracellular matrix; EGF-endothelial growth factor; GMP-guanidine monophosphate; GP-glycoprotein; HMWK-high-molecular-weight kininogen; Ig- immunoglobulin; IP 3 - inositol triphosphate; IP-PGI 2 receptor; MPV-mean platelet volume; P2Y 1 and P2Y 12 - ADP receptors; PAI- 1 - plasminogen activator inhibitor- 1; PAR-protease-activated receptor; PF 4 - platelet factor 4; PGG 2 - prostaglandin G2; PGH 2 - prostaglandin H2; PDCI-platelet-derived collagenase inhibitor; PDGF- platelet-derived growth factor; PECAM- 1 - platelet– endothelial cell adhesion molecule-1; PGI 2 - prostaglandin I 2 (prostacyclin); RGD-arginine-glycine- aspartic acid receptor target; SCCS-surface-connected canalicular system; STR-seven-transmembrane repeat receptor; TGF-β-transforming growth factor-β; TPα and TPβ-thromboxane receptors; TXA 2 - thromboxane A2; VEGF/VPF-vascular endothelial growth factor/vascular permeability factor; VWF-von Willebrand factor INDEX TERMS: Cell adhesion molecules, eicosanoid synthesis, glycoprotein, ligands, prostaglandin, platelet adhesion, platelet aggregation, platelet agonists, platelet count, platelet function, platelet production, platelet secretion, platelet structure Clin Lab Sci 201 5 ;28(2): 125 George A. Fritsma, MS, MLS , The Fritsma Factor, Your Interactive Hemostasis Resource, Fritsma & Fritsma LLC, Birmingham, AL Address for Correspondence: George A. Fritsma, MS, MLS, The Fritsma Factor, Your Interactive Hemostasis Resource, Fritsma & Fritsma LLC, 153 Redwood Drive, Birmingham, AL 35173 , George@fritsmafactor.com Platelets are blood cells that are released from bone marrow megakaryocytes and circulate for approximately 10 days. They possess granular cytoplasm with no nucleus and their diameter when seen in a Wright- stained peripheral blood film averages 2.5 um with a subpopulation of larger cells, 4–5 um. Mean platelet volume (MPV), as measured in a buffered isotonic suspension flowing through the impedance-based detector cell of a clinical profiling instrument, is 8– 10 fL. Circulating, resting platelets are biconvex, although in EDTA blood they tend to “round up.” On a blood film, platelets appear circular to irregular, lavender, and granular, although their diminutive size makes them hard to examine for internal structure.^1 In the blood, their surface is even, and they flow smoothly through veins, arteries, and capillaries. The normal peripheral blood platelet count is 150– 400,000/μL. This count represents only two thirds of available platelets because the spleen sequesters the remainder. In hypersplenism or splenomegaly, increased sequestration may cause a relative thrombocytopenia. Under conditions of hemostatic need, platelets move from the spleen to the peripheral blood and answer cellular and humoral stimuli by becoming irregular and sticky, extending pseudopods, and adhering to neighboring structures or aggregating with one another.

Platelet Structure Platelet Plasma Membrane The platelet plasma membrane is a standard bilayer composed of proteins and lipids (Figure 1). 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 1. 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 and enzymes. 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 A 2 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.2, 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 20–30 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; Figure 2). The SCCS twists sponge-like throughout the platelet, storing additional quantities of the same hemostatic proteins in the glycocalyx. Dense Tubular System Parallel and closely aligned to the SCCS is the dense tubular system (DTS), a remnant of the rough endoplasmic reticulum. The DTS sequesters Ca2+^ and bears enzymes that support platelet activation. These enzymes include phospholipase A 2 , cyclooxygenase, and thromboxane synthetase, which support the eicosanoid synthesis pathway that produces thromboxane A 2.

Table 2. Platelet STR Receptor-Ligand Interactions Coupled to G proteins Receptor Ligand G Proteins PAR1 Thrombin Coupled to G 1 protein that reduces cAMP; coupled to Gq and G 12 proteins that increase IP 3 and DAG PAR4 Thrombin Coupled to Gq and G 12 proteins that increase IP 3 and DAG P2Y 1 ADP Coupled to Gq protein that increases IP 3 and DAG P2Y 12 ADP Coupled to G 1 protein that reduces cAMP TPα and TPβ TXA 2 Coupled to Gq protein that increases IP 3 and DAG α 2 - adrenergic Epinephrine Coupled to G 1 protein that reduces cAMP; potentiates effects of ADP, thrombin, and TXA 2 IP PGI 2 Coupled to GS protein that increases cAMP to inhibit activation STRs are named for their peculiar “seven-transmembranous repeat” 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; IP 3 , inositol triphosphate; PAR, protease-activated receptor; PGI 2 , prostaglandin I 2 (prostacyclin); STR, seven-transmembrane repeat receptor; TXA 2 , thromboxane A2; P2Y 1 and P2Y 12 , ADP receptors; TPα and TPβ, thromboxane receptors; IP, PGI 2 receptor. The cytoplasm also contains intermediate filaments, ropelike polymers 8–12 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. The α-granules are filled with proteins, some endocytosed, some synthesized within the megakaryocyte (Table 3). 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.^4 There are 2–7 dense granules per platelet. Also called dense bodies, these appear later than α-granules in megakaryocyte differentiation and stain black (opaque) when treated with osmium in transmission electron microscopy. Small molecules are endocytosed and are stored in the dense granules (Table 4). Table 3. Representative Platelet α-Granule Proteins Coagulation Proteins Non-coagulation Proteins Proteins Present in Platelet Cytoplasm and α - Granules Endocytosed Fibronectin Albumin Fibrinogen Immunoglobulins Megakaryocyte-synthesized Factor V — Thrombospondin — VWF — Proteins Present in α - Granules But Not Cytoplasm (Megakaryocyte-Synthesized) β-thromboglobulin EGF HMWK Multimerin PAI- 1 PDC Plasminogen PDGF PF4 TGF-β Protein C inhibitor VEGF/VPF Platelet Membrane-Bound Proteins Restricted to α-granule membrane P-selectin GMP — Osteonectin In α-granule and plasma membrane GP IIb-IIIa cap GP IV CD GP Ib-IX-V PECAM- 1 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.

Table 4: Dense-Granule (Dense Body) Contents Small Molecule Comment ADP Nonmetabolic, supports neighboring platelet aggregation by binding to ADP receptors P2Y 1 , P2Y 12 ATP Function unknown, but ATP release is detectable upon platelet activation Serotonin Vasoconstrictor that binds endothelial cells and platelet membranes Ca2+^ and Mg2+^ Divalent cations support platelet activation and coagulation ADP, Adenosine diphosphate; ATP, adenosine triphosphate; P2Y 1 and P2Y 12 , 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 Function Although the following discussion seems to imply a linear and stepwise process, adhesion, aggregation, and secretion, collectively called platelet activation, are often simultaneous.5, Adhesion: Platelets Bind Elements of the Vascular Matrix In arterioles, where the shear rate (viscosity) is over 1000 s-^1 , platelet adhesion and aggregation require a sequence of events that involves collagen, tissue factor, phospholipid, VWF, and a number of platelet receptors, ligands and activators (Figure 3).^7 Normal vascular intimasuppress hemostasis:

Prostacyclin
Heparan sulfate
Tissue factor pathway inhibitor
Nitric oxide
Thrombomodulin Figure 3. Normal blood flow in intact vessels. RBCs and platelets flow near the center, WBCs marginate and roll. Endothelial cells and the vascular intima provide the listed properties that suppress hemostasis. Legend: EC, endothelial cell; FB, fibroblast; lines, collagen; PLT, platelet; RBC, red blood cell; SMC, smooth muscle cell; WBC, white blood cell. Injury to the blood vessel wall disrupts the collagen of the extracellular matrix (ECM).^8 Damaged endothelial cells release VWF from cytoplasmic storage organelles (Figure 4).^9 VWF, whose molecular weight ranges from 800,000 to 2,000,000 Daltons “unrolls” like a carpet, adheres to the injured site, and exposes sites that partially bind the platelet membrane GP Ib-IX-V 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 4. Trauma to the blood vessel wall exposes extracellular collagen and releases von Willebrand factor, triggering platelet adhesion and aggregation. Subendothelial tissue factor and phospholipids support coagulation. Legend: EC, endothelial cell; 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 GPVI comes in contact with the exposed ECM collagen.^10 When type I fibrillar collagen binds platelet GPVI, the receptor triggers internal platelet activation pathways, releasing TXA 2 and ADP.^11 These agonists attach to their respective receptors: TPα and TPβ for TXA 2 , and P2Y 1 and P2Y 12 for ADP, raising the affinity of GP Ia-IIa for collagen. The combined effect of GP Ib-IX-V, GP VI, and GP Ia-IIa causes the platelet to

Figure 5. Eicosanoid synthesis. Ligands (agonists) ADP, thrombin, collagen, or epinephrine bind their respective membrane receptors. The combination activates phospholipase A 2. Phospholipase A 2 releases arachidonic acid from membrane phosphatidyl inositol. Arachidonic acid is acted upon by cyclooxygenase, peroxidase, and thromboxane synthase to produce TXA 2 , which activates the platelet. When reagent arachidonic acid is used as an agonist, it bypasses the membrane and directly enters the eicosanoid synthesis pathway. Legend: ADP, adenosine diphosphate; PgG 2 , prostaglandin G 2 ; PgH 2 , prostaglandin H 2 ; TXA 2 , thromboxane A 2. REFERENCES

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