platelets and the vessel wall

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1380 HEMOSTASIS

In a 1881 communication to the Turin Royal Academy

of Medicine, the Italian physician Giulio Bizzozero dis-

closed the presence in circulating human blood of dis-

crete elements that he termed piastrine (blutplttchen

in a 1882 publication in a German journal and petites

plaques in a communication in French).1 Previously

speculated to be merely nonphysiologic granular aggre-

gates, blood platelets have since become central to ourunderstanding of thrombosis and hemostasis, and detailed

understanding of their participation in cardiovascular

disease, stroke, and even cancer has led to remarkable

progress in the rational treatment of these disorders.

Although platelets are most often studied in the

context of their ability to form a hemostatically effective

plug, it is now widely recognized that their influence

extends far beyond this process to all aspects of hemo-

stasis, as well as to wound healing and vascular remodel-

ing. For example, platelets generate or secrete biologically

active mediators such as thromboxane A2 (TXA2) and

serotonin, which not only amplify platelet activation

responses but also modulate vascular tone. In addition,platelets secrete a broad array of granule constituents

that stimulate vessel repair, induce megakaryocytopoie-

sis, promote coagulation, and limit fibrinolysis.

The same pathways that lead to platelet plug forma-

tion can also produce pathologic thrombosis, a process

that has been described as hemostasis occurring at the

wrong time or in the wrong place. Platelets are particu-

larly important for hemostasis on the arterial side of the

circulation, where blood flows under higher pressure and

experiences greater shear force. As a result, platelet func-

tion is generally considered to be critical to the patho-

genesis of arterial thrombosis and less so for venous

thrombosis, and antiplatelet drugs are most widely usedin the former setting. However, this distinction between

the mechanisms underlying arterial and venous throm-

bosis is not absolute, and the spectrum of thrombotic

disorders should be considered a continuum.

Arterial thrombosis is a particularly common problem

in middle-aged and older adults and is a major cause of

morbidity and mortality in developed countries. The

thrombi that arise in atherosclerotic vessels are predomi-

nantly platelet in origin and are the proximate cause of

myocardial infarction and most cerebrovascular acci-

dents. Although arterial thrombosis is considerably less

common in children than adults, it may contribute to

major morbidity in patients with sickle cell disease, aswell as complications of some childhood infections,

Kawasakis syndrome, and various forms of arteritis,

autoimmune disorders, hemolytic-uremic syndrome,

and thrombotic thrombocytopenic purpura (see Chapter

33).

In this chapter we review platelet structure and func-

tion, with special emphasis on the cell surface glycopro-

teins that function as sentries for areas of vascular damage

and the signal transduction events that both amplify and

limit platelet responsiveness. The information provided

here should be helpful in understanding subsequent

chapters that describe inherited and acquired platelet

disorders (see Chapters 29 and 33) and the role of the

adhesive protein von Willebrand factor (VWF) (see

Chapter 30) in hemostasis. Finally, there is growing

appreciation of the role that platelets play in inflamma-

tion and the pathogenesis of atherothrombosis, which is

briefly discussed at the end of the chapter.

PLATELET MORPHOLOGY ANDSUBCELLULAR ORGANIZATION

Platelets are adhesion and signaling machines that circu-

late as small, disc-shaped cellular fragments in the whole

blood of healthy individuals at a concentration of approx-

imately 150,000 to 300,000/L. Early studies suggestedthat platelets might be produced via cytoplasmic frag-

mentation along a network of internal demarcation

membranes that were observed in large, polyploid mega-

karyocytes.2,3 More recent studies,4-6 however, support

the notion that proplatelets are assembled and packagedwith their various constituents at the ends of long cyto-

plasmic extensions of differentiated megakaryocytes that

have migrated from the proliferative osteoblastic niche to

the capillary-rich vascular niche of the bone marrow

microenvironment,7 with the invaginated demarcation

membrane system serving simply as a reservoir of inter-

nal membrane used for proplatelet extension.8,9 Once

adjacent to the adluminal face of the endothelium,

proplatelets are released into the bloodstream, where

they circulate as mature platelets for approximately 7 to

10 days before being cleared by the liver and spleen 10

their life span being controlled, at least in part, by

an antagonistic balance between the apoptotic proteinsBcl-xL and Bak.11

The size of resting platelets is somewhat variable,

averaging approximately 1.5 m in diameter and 0.5 to1 m in thickness. Platelet size is undoubtedly regulatedby numerous factors during their biogenesis, but both the

224-kd nonmuscle myosin heavy chain IIA (MYHIIA)

and the cell surface glycoprotein Ib (GPIb) complex

appear to play critical roles. Thus, mutations in the

MYH9 gene predominantly interfere with contractile

events important for platelet formation,12whereas failure

to express GPIbthe molecular basis for the platelet

disorder known as Bernard-Soulier syndrome13,14dis-

rupts critical associations with the cytoskeletal proteinfilamin15,16 that play an essential role in both platelet

formation and platelet compliance.17In both these inher-

ited platelet disorders, platelets can appear as large as

lymphocytes (see Chapter 29). Correction of GPIb

expression in GPIb-deficient (Bernard-Soulier) mice has

been shown to restore platelets to their normal size.18

The volume of a platelet (mean platelet volume)

normally ranges from 6 to 10 fL (1 fL =1015L). Plateletdensity is also variable,19and the issue of whether young

platelets are more20,21or less22dense as they gain versus

lose content during their circulating lifetime has never


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Chapter 25 Platelets and the Vessel Wall 1381

been satisfactorily resolved. Because platelets retain most

species of messenger RNA (mRNA) for a short period

after their release from bone marrow megakaryocytes,23

young platelets can be distinguished from older ones by

their RNA content.24

As shown in Figure 25-1A and C, resting platelets are

discoid in shape, largely because of the presence of a cir-

cumferential coil of microtubules,25,26

and they are packedwith numerous electron-opaque alpha granules, a few

dense granules (granule contents and their functions are

discussed later), several mitochondria, and lysosomes.27

Platelets also retain a few Golgi remnants, as well as occa-

sional vestiges of rough endoplasmic reticulumthe

exception being platelets from patients with rapid platelet

turnover, in whom very young platelets containing more

abundant protein synthesis machinery are readily observed

in the circulation. Platelets also contain two highly spe-

cialized membrane systems not found in other cells of the

body: the surface-connected open canalicular system

(OCS) (see Fig. 25-1B and C) and the dense tubular

system (DTS). The OCS is a series of tortuous invagina-tions of the plasma membrane that appear to tunnel

throughout the cytoplasm of the cell28 and serve as an

internal reservoir of plasma membrane that is called upon

when platelets round up, extend lamellipods and filopods

(see Fig. 25-1B and D), and spread during platelet activa-

tiona process that can increase the surface area of

exposed plasma membrane by more than 400%.29Because

OCS channels are proximal to internal granules, they also

probably function as a conduit for the rapid expulsion of

alpha and dense granule contents during platelet activa-

tion.30The DTS, on the other hand, is a remnant of the

smooth endoplasmic reticulum31and is found randomly

dispersed throughout the cytoplasm. The DTS appears to

be one of several organelles within the platelet known to

harbor high concentrations of calcium,32,33

and it isthought to contain a 100-kd calcium adenosine triphos-

phatase (ATPase) known as SERCA2b34that functions to

sequester and store cytosolic calcium in resting cells.

Recent evidence suggests that adenosine diphosphate

(ADP) is able to induce selective release of calcium from

the DTS35whereas activation of the GPIb/V/IX receptor

for VWF releases calcium primarily from a poorly defined

acidic compartment36 within the cell.35 Thrombin, a

strong platelet agonist, appears to elicit release of calcium

from both stores on binding to the platelet thrombin

receptor PAR1.35

The platelet cytoskeleton is composed of a single

rigid, but dynamic microtubule approximately 100 m inlength that is coiled about 8 to 12 times around theequatorial plane of the cell.37-39This marginal band of

microtubules is largely responsible for maintaining the

discoid shape of the resting cell, as illustrated by the

observations that (1) incubation of platelets with colchi-

cinean agent that dissolves microtubulesresults

in their rounding,40 (2) platelets from mice lacking

A

B

C D

FIGURE 25-1.Platelet morphology. Resting platelets (shown in thin section in Aand from a scanning electron micrograph of a flash-frozen,freeze-dried platelet in C) are shaped like a disc and contain numerous electron-opaque alpha granules, a few dense granules, and several mito-

chondria and lysosomes. A circumferential coil of microtubules (mchighlighted with an oval) is responsible for maintaining their discoid shape

Platelets also contain a number of cytoplasmic membrane systems that subserve specialized functions, including vestiges of the smooth endoplasmic

reticulum that sequester calcium and tortuous invaginations of the plasma membrane that form a surface-connected open canalicular system

(OCS). When platelets become activated (Band D), they rapidly round up, extend lamellipodia (lam) and filopodia (fil), and release the contents

of their granules, often into the nearby OCS. (Photographs generously provided by John H. Hartwig and used with permission.)


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1382 HEMOSTASIS

1-tubulin remain largely spherical,41 and (3) plateletspherocytosis in humans results when tubulin fails to

polymerize normally into microtubules.42Directly under-

neath the plasma membrane lies an intricate, two-dimen-

sional, tightly woven membrane skeleton43composed of

nonerythroid spectrin,44,45a network of actin filaments,43,45

vinculin,46and the actin-binding protein filamin,47which

itself is tethered to the inner face of the plasma mem-brane via linkages with the cytoplasmic domain of

GPIb.15,47The membrane skeleton, because of its loca-

tion, serves as a scaffold that links elements of the plasma

membrane with contractile elements of the cytoskeleton

and cytosolic signaling proteins and thereby regulates

such diverse functions as receptor mobility,48,49receptor

clustering,50-52 and signal transduction.53 Finally, the

platelet is filled with an extensive cytoplasmic network of

actin filaments45,54 organized by the actin-binding pro-

teins filamin55,56 and -actinin57 that constitute itscytoskeleton.

When platelets become exposed to components of

the extracellular matrix58

or to soluble agonists such asADP59or thrombin,60,61they undergo dramatic changes

in their morphology.62,63The marginal band of microtu-

bules disappears,54which allows the platelet to transform

from a disc to an irregular sphere. At nearly the same

time, the actin filamentcapping protein -adducinbecomes phosphorylated and dissociates from existing

F-actin filaments,64thereby exposing the barbed end of

the filament to cytosolic actin monomers and driving

rapid polymerization of actin into microfilaments.62This

has the dual effect of driving the extension of lamellipo-

dia and filopodia and forcing granules toward the center

of the platelet, where they can fuse with membranes of

the OCS and release their contents to the exterior of thecell. Phosphorylation of myosin additionally induces

contractile events that facilitate centralization of the

granules.65

PLATELET GENOMICS AND PROTEOMICS

Though anucleate, platelets contain measurable and

manipulable levels of megakaryocyte-derived mRNA,23

at least some of which is capable of being synthesized

into small, but detectable amounts of protein.66,67Both

serial analysis of gene expression (SAGE) and gene

microarray analysis have been used to estimate the sizeand composition of the platelet transcriptome.68-70A con-

sistent finding of all genomic analyses performed to date

is that mitochondrially derived transcripts dominate the

platelet transcriptomepresumably because of persis-

tent transcription of the mitochondrial genome after

platelet release from the bone marrow. This problem has

recently been addressed by analyzing the transcriptome

of cultured megakaryocytes derived from cord blood

stem cells.71Of the 20,488 genes present in the human

genome, 2000 to 3000 distinct transcripts have been

identified in unstimulated plateletsconsiderably fewer

than normally found in a nucleated cell, but perhaps

more than one might have expected from an anucleate

circulating cellular fragment. One of the more surprising

findings in recent years has been the identification of

heterogeneous nuclear RNA (hnRNA) in the platelet

cytosol, as well as all of the spliceosome components

necessary to splice the hnRNA into mature message that

can thereafter be translated into protein.72

Enlisted duringthe activation process, signal-dependent protein transla-

tion has thus far been demonstrated for mRNA mole-

cules encoding interleukin-1 (IL-1),72 tissue factor,73and Bcl-3,74 the protein products of which have the

potential to influence inflammation, thrombosis, and

wound repair.

The platelet proteome appears to be equally complex

and diverse and, unlike the transcriptome, reports both

the breadth and relative amounts of protein products

actually present in the cell. Obtained by refined two-

dimensional gel electrophoretic techniques that were

originally developed in the 1970s75,76or by liquid chro-

matographic separation, proteins are fragmented andseparated via a combination of proteolytic and ionization

techniques and then analyzed by mass spectrometry.

Such analysis has allowed the identification of dozens of

proteins present in complex cellular lysates or subcellular

fractions (see elsewhere77,78 for recent reviews of this

topic). In addition to yielding the expected menu of

major plasma membrane glycoprotein receptors, one of

the more complete global profiling analyses to date79

identified a core platelet proteome composed of 641 pro-

teins, including an abundance of molecules involved in

signal transduction, cytoskeletal change, and metabo-

lismunderstandable given the importance of cellular

activation and its control in platelet function. By combin-ing prefractionation methods with suitable separation

techniques, proteomic analysis has also been used to

compile an inventory of proteins that are either (1) post-

translationally modified (normally by phosphorylation)

during the platelet activation process80-82or (2) present

at low abundance in the total platelet proteome but

enriched within various subcellular compartments,

including the platelet cytoskeleton,83alpha granules,81,84,85

membrane fraction,86 membrane rafts,87 and

microparticles.88

ANTITHROMBOTIC COMPONENTSOF THE VESSEL WALL

Although hundreds of thousands of platelets per micro-

liter circulate in blood, under normal conditions very few,

if any, interact with the intact vessel wall because the

endothelial lining of the blood vessel presents an excel-

lent nonthrombogenic surface. In fact, this property of

the vessel wall has not yet been duplicated in any pros-

thetic or extracorporeal device. Healthy endothelium not

only provides an effective barrier between blood compo-

nents and the highly thrombogenic components of the


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subendothelium (see later) but also actively produces

both membrane-bound and secretory products that limit

fibrin generation and promote clot dissolution. For

example, heparin-like glycosaminoglycans present on the

luminal side of the endothelial cell surface recruit plasma

antithrombin, which effects a conformational change that

promotes binding and neutralization of thrombin and

other serine proteases.89

Thrombin, when bound to theendothelial cell surface receptor thrombomodulin, takes

on anticoagulant properties via its cleavage and activation

of protein C, which in turn cleaves coagulation factors V

and VIII, thereby further suppressing thrombin genera-

tion.90Endothelial cells also express a specific receptor

for activated protein C that serves to concentrate the

protein on the endothelial surface (see Chapter 26).91

Finally, endothelial cells synthesize, secrete, and rebind

tissue plasminogen activator,92,93 which activates plas-

minogen to facilitate fibrin dissolution (see Chapter 27).

These activities are summarized in schematic form in

Figure 25-2.

The endothelial cell also produces two importantinhibitors of platelet activation: prostacyclin (PGI2)

94-96

and nitric oxide (NO).97-99A labile oxygenated metabolite

of arachidonic acid generated by endothelial cell cyclo-

oxygenase-2 (COX-2), PGI2diffuses out of the cell and

binds to a platelet Gsproteincoupled receptor (GPCR)

known as the isoprostenoid (IP) receptor.100,101 Such

binding stimulates adenylate cyclase to increase cytosolic

cyclic adenosine monophosphate (cAMP) levels, which

(1) activates a pump in the DTS that decreases cytosolic

Ca2+, thereby helping keep platelets quiescent, and (2)

activates protein kinase A (PKA), the actions of which

will be discussed later. PGI2also has potent vasodilatory

effects by binding to IP on arterial smooth muscles cellsto effect vessel relaxation.94 The PGI2 produced by

vascular endothelium thus serves to counterbalance

the proaggregatory and vasoconstrictor activities of the

platelet-derived prostanoid TXA2, the biology of which is

discussed later. In fact, upsetting the delicate balance

between COX-1derived TXA2 and COX-2derived

PGI2 has been shown to increase the risk for adverse

cardiovascular events.102

Whereas PGI2stimulates adenylate cyclase to produce

cAMP, NO, a product of -arginine generated by endo-

thelial nitric oxide synthase (eNOS),103directly activates

platelet guanylate cyclase, which results in increased cyto-

solic levels of cyclic guanosine monophosphate (cGMP).Although platelet responses to low levels of this cyclic

nucleotide can at first be mildly stimulatory,104 cGMP,

largely via its activation of protein kinase G (PKG), has

the overall effect of dampening platelet responses, inhibit-

ing platelet adhesion105,106 and aggregation,107-110 and

cAMP

CD39

EPCR

TM

COX-2eNOS

NO PGI2

GAGs

APC

ATIII Plasminogen

AMP

Plasmin

FVon, FVIIIon

ADP Fvoff, FVIIIoff

thrombin

PC

TPA

Adenylatecyclase

Guanylatecyclase

Gs

Gs

Dense tubularsystem

PKG

Platelet

Endothelium

Multiple inhibitory signaling pathways

PKA

SERCA2b Ca2+Ca2+

Ca2+

Ca2+ Ca2+Ca2+Ca

2+Ca2+

Ca2+Ca2+

cGMP

FIGURE 25-2. Anticoagulant and antithromboticcomponents of the vascular endothelium. Endothelial

cells produce a number of substances, including nitric

oxide (NO) and prostacyclin (PGI2), that act on plate-

let surface receptors to dampen platelet responsiveness.

They also scavenge the platelet agonist adenosine

diphosphate (ADP), inactivate thrombin, and activate

the fibrinolytic enzyme plasmin. APC, activated protein

C; AMP, adenosine monophosphate; ATIII, antithrom-

bin III; cAMP, cyclic adenosine monophosphate;

cGMP, cyclic guanosine monophosphate; COX-2,

cyclooxygenase-2; eNOS, endothelial nitric oxide syn-

thase; EPCR, endothelial cell protein C receptor; FV,factor V; GAGs, glycosaminoglycans; TM, thrombo-

modulin; IP, isoprostenoid; PC, protein C; PKA,

protein kinase A; PKG, protein kinase G; TPA, tissue

plasminogen activator.


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1384 HEMOSTASIS

impeding platelet-mediated recruitment of leukocytes

during the inflammatory response.111 Its mechanism of

action is discussed in more detail later.

In addition to the soluble metabolites PGI2and NO,

endothelial cells also express on their surface a potent

adenosine diphosphatase (ADPase) known as CD39 that

scavenges plasma ADP to prevent platelet aggrega-

tion.112,113

Finally, it is important to note that inflamma-tory cytokines, oxidized lipids, and immune complexes

can, under pathologic conditions, inhibit these protective

biochemical pathways and impair the antithrombotic

state of the endothelial cell. The latter changes permit

unrestrained formation of platelet- and fibrin-containing

thrombi, as well as thrombus formation beyond sites of

vascular injury, and can thus contribute to atherothrom-

bosisa topic that is discussed more extensively at the

end of this chapter.

REACTING TO THE BREACHCELL SURFACE

RECEPTORS THAT MEDIATE TETHERINGAND ADHESION AND TRANSMIT EARLYACTIVATION SIGNALS

As antithrombotic as the endothelial lining is, the under-

lying extracellular matrix consists of a rich mixture of

glycosaminoglycans into which are embedded an abun-

dance of highly concentrated prothrombotic proteins,

including structural components such as collagen and

elastin (which constitute 30% of body weight) andadhesive proteins such as laminin, fibronectin, and VWF.

Not surprisingly, platelets have evolved receptors for

most of these proteins and initiate a series of rapid bio-

chemical events both on the surface and inside the cellwhen exposed to them. As a result, adhesion is an activat-

ing event!

The large number of circulating red blood cells serve

to marginate platelets, and when the vessel wall is

breached, either by mechanical injury or after rupture

of atherosclerotic plaque, the first layer of platelets to

encounter exposed matrix undergoes a series of sequen-

tial events similar to what leukocytes experience during

the inflammatory responsenamely, tethering, initial

signaling to the cell interior, integrin-mediated adhesion,

and cytoskeletally directed cell spreading. Whereas leu-

kocyte tethering is mediated by members of the selectin

family, the first layer of platelets become tethered onVWF,114which is sprinkled on exposed collagen fibers.

VWF interacts with a high-affinity, platelet-specific mul-

tisubunit receptor known as the GPIb/V/IX complex.115

This latter complex, which is expressed at approximately

25,000 copies per cell,116 binds to the A1 domain of

VWF117with high-enough affinity to tether platelets even

under conditions of arterial shear.118Loss of the GPIB/

V/IX receptor in both humans and mice results in a clini-

cal condition known as Bernard-Soulier syndrome,13,14

which is characterized not only by an increase in platelet

size but also by prolonged bleeding caused, in large part,

by the inability of platelets to adhere to the vessel wall.

After engagement with its ligand, GPIb acts through

membrane-proximal Src family kinases,119 through

adapter molecules,120and to a lesser extent, via its asso-

ciation with immunoreceptor tyrosinebased activation

motif (ITAM)-bearing subunits121,122 to transmit early

activation signals123,124that together result in the recruit-

ment and activation by tyrosine phosphorylation of phos-pholipase C2 (PLC2),125,126a key enzymatic componentof platelet amplification that is required to achieve throm-

bus growth and stability (Fig. 25-3).127

Once tethered, two different platelet integrinseach

of which exists in a low-affinity state on the platelet

surfacebegin to engage specific extracellular matrix

components and, together with the small calcium tran-

sients and kinase-generated signals emanating from the

GPIb complex and from the mechanical shear force gen-

erated by the flowing blood,128initiate the reciprocal pro-

cesses of platelet adhesion and activation. Thus, the 21integrin binds to exposed collagen fibrils,129whereas the

integrin receptor 61 engages laminin.130

Both theseintegrins hand off to a member of the immunoglobulin

superfamily, GPVI,131,132which via its noncovalent asso-

ciation in the plane of the plasma membrane with the

ITAM-bearing Fc receptor chain dimer131,133 elicitsstrong PLC2-dependent events that (1) begin theprocess of cytoskeletally directed shape change and

cell spreading (discussed earlier); (2) initiate signal trans-

duction pathways (illustrated in Fig. 25-3) that cause

dramatic structural changes in platelet integrins and

thereby result in their adopting a high-affinity, ligand-

bindingcompetent conformation134a process known

as inside-out signal transduction (to be described in

more detail later); and (3) facilitate fusion of alpha anddense granules with the OCS and underlying plasma

membrane.

PLATELET GRANULES AND THEIRROLE IN HEMOSTASIS

Platelet-specific granules are synthesized, assembled, and

packaged during megakaryocyte biogenesis, and at later

stages of maturation they appear to come into contact

with microtubules, which then transport them, via the

microtubule motor protein kinesin, along the shafts of

proplatelets until they reach the proplatelet tips.135

Onceinside a mature platelet, platelet granules remain rela-

tively evenly dispersed throughout the cytoplasm, their

contents awaiting threshold signals for cellular activation,

at which time their membranes fuse with the plasma

membrane or, more likely, the invaginated subdomains

of the plasma membrane known as the OCS136 (see

earlier). The regulated secretion of granule contents

ensures that hemostasis remains highly localizedan

event that has recently been exploited to deliver non

platelet-derived procoagulant proteins such as factor VIII

to sites of vascular injury.137,138


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Collagen

Laminin

VWF

GPVI

IT AM

Fyn/Lyn

Syk

PLC2

IT AM

SH2

SH2

SH2

SH2

Granule secretion

Rap1 Integrin activation

Cytoskeletal rearrangementsShape change

Integrin clustering

GPIb-V-IX

FcR

CRP

PI3KSrc

Ca2+

release from DTSIP3+

DAG

PKC

CalDAG-GEF

Syk

Akt2GSK

3

RIAM

Talin

21

LAT,S

LP-76

61

Kinase

Kinase

PI3K,

Ca2+

S S

Gads,B

tk

Gads,B

TKLAT,SL

P-76

FIGURE 25-3. Platelet adhesion receptors that signal through phospholipase C2 (PLC2). Each of the cell surface receptors shown recognizesdifferent component of the extracellular matrix and thus works in coordinated fashion to send early activation signals into the cell. Binding of

platelets to von Willebrand factor (VWF) slows platelets down so that integrins can associate productively with matrix collagen and laminin. Signals

emanating from each of these events are transmitted into the cell interior, in part via the action of receptor-associated Src family kinases (Src, Fyn,

Lyn), which phosphorylate tyrosine residues within nearby immunoreceptor tyrosinebased activation motifs (ITAMs), thus forming a nucleation

point for the assembly of miniature organelles sometime referred to as signalosomes. Signalosomes are themselves composed of the adaptor proteins

LAT, SLP-76, and Gads and the receptor tyrosine kinase Btk and function to localize, phosphorylate, and then activate PLC2, which coordinatesall these responses by generating the classic signaling molecules 1,4,5-inositol triphosphate (IP 3) and diacylglycerol (DAG). Details regarding the

molecular events that take place after the generation of IP3and DAG are shown in Figures 25-4 and 25-6. Btk, Brutons tyrosine kinase; CRP, C-

reactive protein; DTS, dense tubular system; GP1b, glycoprotein Ib; GSK3, glycogen synthase kinase 3; PI3K, posphatidylinositol-3-kinasePKC, protein kinase C; RIAM, Rap1GTP-interacting adaptor molecule; SH2, Src homology domain 2.

Platelets harbor three distinct types of granules (Box

25-1). Twoalpha and dense granulesare found only in

platelets, whereas lysosomes are present in nearly all cell

types. Alpha granules are by far the most numerous, with

as many as 40 to 80 per cell, and they contain a wide array

of proteins and bioactive peptides. For ease of discussion,

Box 25-1 classifies alpha granule proteins as those that

reside within the alpha granule membrane (P-selectin

being the most diagnostic), those pinocytosed from

plasma and packaged (IgG, fibrinogen, albumin),139and

those synthesized by megakaryocytes and stored (VWF,

platelet factor 4, thrombospondin). mRNA molecules

encoding the latter group have all been identified in the

platelet cytoplasm. Upon platelet activation, granules

become redistributed toward the center of the cell,136at

which time SNARE (solubleN-ethylmaleimidesensitive

attachment protein receptor) proteins within the alpha

granule membrane facilitate fusion,140with members of

the Rab family of low-molecular-weight guanosine tri-

phosphatases (GTPases) playing a prominent role in

vesicle docking and exocytosis.141,142 After membrane

fusion, P-selectin143-145 and alpha granule membrane


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1386 HEMOSTASIS

that contain the vasoconstrictive substance seroto-

nin,160-162adenine and guanine nucleotides such as ADP

and ATP, inorganic pyrophosphates163 and polyphos-

phates,164and the divalent cation calcium.165Complexes

of the latter two are probably responsible for the dark

appearance of these bodies on thin-section electron

microscopy.166Dense granule membranes contain a few

components in common with lysosomal membranes, suchas granulophysin (CD63, lysosome-associated membrane

protein-3 [LAMP-3])167and LAMP-2,168as well as mem-

brane proteins also present in alpha granule membranes,

such as P-selectin,167 thus suggesting a common origin

during biogenesis. Like their alpha granule counterparts,

these dense granule membrane proteins become expressed

on the platelet cell surface after granule fusion and secre-

tion and can be used as platelet activation markers. Curi-

ously, a number of plasma membrane glycoproteins,

including GPIb and GPIIb-IIIa, have also been reported

in dense granule membranes.169Dense granule contents,

especially ADP,170play a physiologically important role in

hemostasis, as evidenced by characteristic platelet func-tion defects in patients whose platelets lack dense gran-

ules or their contents,171 collectively known as storage

pool disorders.172-174 Chdiak-Higashi and Hermansky-

Pudlak175syndromes are two such examples of autosomal

recessive dense granule defects that lead to platelet dys-

function and bleeding, the former being associated with

immunodeficiency and the latter with albinism (see

Chapter 29).

Primary lysosomes are the third organelle whose

contents are secreted upon platelet activation, but only

three or fewer per cell are normally identifiable.176

Although a clear role for lysosomes in platelet function

has not been identified, they do contain more than adozen different acid hydrolases, cathepsins D and E, and

other degradative enzymes that can be secreted if plate-

lets are subjected to strong agonist stimulation. Their

contents have been shown to be mildly reduced in the

platelets of individuals with GPS,177in keeping with the

notion that the latter disorder is caused by a defect in

packaging. The membrane proteins on platelet lysosomes

are typical of lysosomes in other cells and include LAMP-

1,178LAMP-2,168and LAMP-3.167

FEED-FORWARD AMPLIFICATION

PATHWAYS INVOLVED IN PLATELETRECRUITMENT AND THROMBUS STABILITY

Although platelet adhesion, early activation signals, and

granule release are prerequisites for thrombus formation,

efficientrecruitment of additional platelets to the site of

the vascular lesion to yield a stable platelet plug requires

a host of additional receptor/ligand interactionseach of

which results in signal transmission and subsequent bio-

chemical and cell biologic changes that help sustain

platelet activation. Among the most important of these is

the binding of released ADP to one of its two platelet G

Box 25-1 Platelet Granules and Their Contents

ALPHA GRANULES

Membrane proteins enriched in the granule membrane: P-

selectin, TLT-1, CD40 ligand (which is cleaved after

exposure on the platelet surface to release soluble

CD40L), and tissue factor.

Membrane proteins present at similar concentrations as they arein the plasma membrane: GPIIb-IIIa, GPIb, PECAM-1,

and perhaps many others

Granule contents:

Synthesized by megakaryocytes: Thrombospondin, VWF,

platelet factor 4, -thromboglobulin, PDGF Endocytosed from plasma or origin not determined:

Albumin, fibrinogen, fibronectin, IgG, Gas6,

coagulation factor V, and many chemokines and growth

factors, including RANTES, bFGF, EGF, TGF-, andVEGF

DENSE GRANULES

ADP, ATP, 5-HT, Ca2+, polyphosphate

LYSOSOMES

Acid hydrolases, elastase, cathepsins, and other

degradative enzymes

ADP, adenosine diphosphate; ATP, adenosine triphosphate;

bFGF, basic fibroblast growth factor; EGF, epidermal growth

factor; Gas6, growth arrestspecific gene 6; GP, glycoprotein;

5-HT, 5-hydroxytryptamine; PDGF, platelet-derived growthfactor; PECAM-1, platelet endothelial cell adhesion molecule-

1; RANTES, regulated on activation, T cell expressed and

secreted; TGF-, transforming growth factor ; TLT-1, TREM(triggering receptor expressed on myeloid cells)-like transcript-

1; VEGF, vascular endothelial growth factor; VWF, von

Willebrand factor.

specific proteins such as TLT-1146become expressed on

the platelet surface, and the contents of the granule are

released into the plasma milieu. Exposed P-selectin, diag-

nostic of an activated platelet,147,148serves to recruit leu-

kocytes to the site of injury,149one of a number of impor-

tant links between thrombosis and inflammation150

(discussed at the end of this chapter). Proteins secreted

from platelets include the adhesive ligands VWF and

fibrinogen, which serve to support platelet-platelet inter-

actions; growth factors and cytokines, which promote cell

migration151and wound healing152and maintain vascular

integrity153

; and autocrine factors such as growth arrestspecific gene 6 (Gas6)154and CD40L,155which are released

and rebind platelet receptors to help amplify platelet

responsiveness. Finally, alpha granules and their contents

are a source of procoagulant proteins, with release of

factor V156and exposure of tissue factor73,157promoting

localized fibrin deposition at sites of vascular injury. Plate-

let alpha granules, or at least their contents,158are severely

reduced in an inherited bleeding disorder known as gray

platelet syndrome (GPS) (see Chapter 29).159

Dense granules (four to eight per platelet) are mor-

phologically distinct, electron-opaque storage organelles


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as a result of the action of phospholipase-generated 1,4,5-

inositol triphosphate (IP3, discussed in more detail

later)to remain cytosolic. Bioavailable calcium ions,

in turn, support a host of additional cellular events,

including more robust granule secretion, activation of

metal iondependent proteases, and activation of cell

surface integrins. The importance of ADP in amplifying

platelet responses is illustrated by the clinical effective-ness of ticlopidine and clopidogrelwidely used phar-

macologic agents that antagonize the activity of P2Y12

in pacifying platelet reactivity and inhibiting platelet

aggregation.181

In addition to ADP-induced, P2Y12-mediated signal-

ing, nearly a dozen other soluble ligands are either gener-

ated or released at sites of vascular injury and function

in signal amplification and platelet activation. These

ligands can, for the sake of simplicity, be broken into

three classes according to the type of platelet receptor to

which they bind (Fig. 25-5). The first class of ligands is

composed of ADP, thrombin, TXA2, and serotonin (5-

hydroxytryptamine [5-HT]), each of which binds to aspecific GPCR that is coupled to the heterotrimeric subunit, Gq. Thus, ADP binds to P2Y1,

182-185thrombin to

the protease-activated receptors PAR1 and PAR4,186,187

TXA2to the thromboxane receptor,188,189and serotonin

to 5-HT2A.190When released as a consequence of ligand

binding to any of these GPCRs, the Gqsubunit binds to

the isoform of phospholipase C (PLC). PLCs are lipidhydrolases that act on membrane-associated phosphati-

dylinositol 4,5-diphosphate (PIP2) to produce the second

messengers IP3and diacylglycerol (DAG). IP3binds and

opens calcium channels, whereas DAG activates the most

abundant forms of protein kinase C (PKC), thereby

epi

2A P2Y12Adenylatecyclase

cAMP Many other cellbiologic effects

Dense tubularsystem

SERCA2b

Granulesecretion

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+Ca2+

Gi2

ADP

Gz Gi2Gz

FIGURE 25-4. Gi proteincoupled receptors on platelets inhibitadenylate cyclase and lower cyclic adenosine monophosphate (cAMP)

levels. Shown are the two major receptors responsible for dampening

the activity of adenylate cyclase. As cAMP levels drop, the ability of the

SERCA2b calcium pump to sequester cytosolic calcium ions is

impaired, thereby allowing calcium-mediated activation events to occur

more readily.

Ca2+

releasefrom DTS

IP3+

DAGPKC

Granule secretion

Cytoskeletal rearrangementsShape change

Integrin clustering

Rap1 RIAM

Talin

CalDAG-GEF

Rap

1

Integrinactivation

PLCPLC

Gq

Gq

Integrins

Src

Kinase

SH2

Syk

ITAMSH2

ADPThrombin

TXA25'HT

Fg, VWF, Collagen,sCD40L, LM, FN

Gas6Ephrins

GPCR RTK

LAT,

SLP-7

6

Gads,

Btk

FIGURE 25-5.Agonist receptors that initiate or amplifyplatelet activation responses (or both). Three different

families of receptors are involved in signal amplification

pathways: Gq-coupled GPCRs, integrins, and receptor

tyrosine kinases (RTKs). Note how each activates either

the or isoform of phospholipase C (PLC). The sumtotal of PLC-generated products serves to determine the

activation state of the platelet, its ability to respond to

vascular injury, and its participation in thrombus growth.

ADP, adenosine triphosphate; Btk, Brutons tyrosine

kinase; DAG, diacylglycerol; DTS, dense tubular system;

GPCR, Gs proteincoupled receptor; 5-HT, 5-hydroxy-tryptamine; IP3, 1,4,5-inositol triphosphate; ITAM, immu-

noreceptor tyrosinebased activation motif; PKC, protein

kinase C; RIAM, Rap1GTP-interacting adaptor molecule;

SH2, Src homology domain 2; TXA2, thromboxane A2;

VWF, von Willebrand factor.

proteincoupled receptors, P2Y12.179,180Like the 2recep-

tor for epinephrine, P2Y12is coupled to an inhibitory G

protein that slows down the activity of adenylate cyclase,

thus lowering cytosolic levels of cAMP (Fig. 25-4). This

greatly potentiates platelet responses by other agonists

because it allows calcium ionsreleased from the DTS


10/20

1388 HEMOSTASIS

initiating additional signaling cascades downstream of

this serine/threonine kinase. As shown in Figure 25-5, it

is the sum of these productsgenerated by the 2 isoformof PLC in response to adhesion and by the isoform ofPLC in response to ligand-GPCR interactionsthat the

platelet integrates when deciding whether to become

fully activated. This concept is important in the context

of designing pharmacologic strategies to inhibit plateletfunction because blocking adhesion and its consequent

activation of PLC2 leaves PLC-mediated platelet acti-vation largely intact, and vice versa.

The second class of signal amplifiers consists of the

cell surface integrins themselves.191As illustrated in the

middle section of Figure 25-5, when ligands bind to

integrins, the Src family kinases associated with integrin

cytoplasmic tails192trigger a series of incompletely under-

stood amplification events193that have been collectively

termed outside-in signaling.194-196Although this has best

been demonstrated after interaction of the major platelet

integrin IIb3 with its ligand fibrinogen, signals also

probably emanate from 21134,197,198

and 61132

uponengaging collagen and laminin, respectively. Activation

signals from the latter two may be relatively weak by

comparison because of the fact that only a few thousand

of each are expressed on each platelet as compared with

50,000 to 80,000 IIb3 receptors per cell.199,200 Theprotein kinases Syk and FAK have been shown to become

activated downstream of IIb3engagement, as has activa-tion of PLC2.201 However, the details of these eventsremain to be worked out. Finally, there is at least one

autocrine loop that uses integrin-mediated outside-in

signal amplificationthat being cleavage and rebinding

of soluble CD40L after alpha granule fusion and

secretion.155

The third class of feed-forward amplification reac-

tions is mediated by ligand-activated plasma membrane

receptor tyrosine kinases. The first of these to be described

were receptors for Gas6, a vitamin Kdependent protein

related to the anticoagulant protein S. Gas6 is thought

to reside in platelet alpha granules202,203and, like other

alpha granule proteins, becomes secreted upon platelet

activation. Interestingly, platelets have three different

receptors for Gas6Axl, Sky, and Merall of which

have active cytoplasmic tyrosine kinase activity (see Fig.

25-5). Upon engagement, Gas6 receptors appear to be

able to trigger tyrosine phosphorylation of the 3integrin

cytoplasmic domain and thereby support outside-in inte-grin signaling, as well as activate phosphatidylinositol-

3-kinase (PI3K) to further sustain granule secretion.154Platelets also express two members of the Eph receptor

tyrosine kinase family, EphA4 and EphB1, which when

in contact with their membrane-bound counter-receptor

Ephrin B1, stimulate tyrosine phosphorylation of the

integrin 3tail and activate the integrin activator Rap1b.204Like Gas6 signaling,154,205,206 genetic loss or pharmaco-

logic blockade of Ephrin/Eph kinase interactions results

in decreased ability to form a stable thrombus or retract

a fibrin clot.207

ACTIVATION OF THE MAJOR PLATELETINTEGRIN aIIBb3(GPIIB-IIIA COMPLEX)THEFINAL COMMON END POINT OF PLATELETACTIVATION

Human platelets express at least five different members

of the 24-member integrin family,196,208 including three

1 integrins (21, 51, and 61specific for collagen,fibronectin, and laminin, respectively) and two 3integ-rinsv3and its close relative IIb3(also known as theGPIIb-IIIa complex). IIb3is by far the most abundantand well studied. This section focuses on our current

understanding of how IIb3becomes transformed froma resting to an active ligand-bindingcompetent confor-

mation, with the understanding that the biochemical and

cell biologic principles described for this integrin may

well apply to the others.

As shown in schematic form on the left side of Figure

25-6, IIb3exists on the platelet surface in a bent-overconformation that is unable to associate effectively with its

major soluble ligands fibrinogen, VWF, and fibronec-tin.209,210Though relatively short, the cytoplasmic domains

of IIband 3are thought to play a key role in maintainingthe off state of this integrin complex as a result of weak

charge interactions between them211,212 that allow the

hydrophobic transmembrane domain helices of each

subunit to interact and maintain the integrin in a low-

affinity state.213,214 When platelets become activated

either by adhesion- or soluble agonist-mediated

eventscalcium and DAG, generated as a result of the

actions of PLC2 and PLC(see Figs. 25-3 and 25-5),bind to and activate PKC and the guanine exchange factor

CalDAG-GEF1. As illustrated in Figure 25-6, each of

these can independently activate Rap1215-219

a low-molecular-weight GTPase that has been implicated in

integrin activation.220-222Rap1 appears to activate integrins

via an effector molecule known as RIAM (Rap1GTP-inter-

acting adaptor molecule), which recruits the highly abun-

dant cytosolic protein talin to the inner face of the plasma

membrane to form an integrin activation complex. Binding

of RIAM-associated talin to the 3integrin subunit repre-sents the final common step in integrin activation223-225

because it disrupts the weak ionic clasp between the IIb3tails and thereby allows tail separation and a dramatic,

rapid unfolding of the extracellular domain.210Simultane-

ous conformational changes in the integrin head226,227

result in the formation of an integrin receptor with highaffinity for its soluble adhesive ligands. Finally, clustering

of integrins occurs228and ensures that bound ligands effec-

tively broker with high avidity the platelet-platelet interac-

tions that permit thrombus growth and stabilization.

CELL SURFACE AND CYTOSOLIC PROTEINSTHAT LIMIT PLATELET RESPONSES

As anyone who has suffered a myocardial infarction or

thrombotic stroke can attest to, unrestrained thrombus


11/20


growth at inappropriate sites can be as harmful as exces-

sive bleeding because it can result in vessel occlusion,

ischemia, and tissue damage. Numerous active processes

are therefore in place to limit platelet responsiveness in

healthy vessels so that thrombus growth is kept localizedto specific sites of vascular injury and dissolution of the

platelet plug during recovery is facilitated.

As discussed earlier, healthy endothelium contrib-

utes to platelet passivation via rather continuous genera-

tion of PGI2and NO, which act on platelets by activating

adenylate and guanylate cyclases to increase intracellular

levels of cAMP and cGMP. These messengers activate

PKA and PKG, respectively (illustrated in Fig. 25-2).

PKG controls the threshold for platelet activation pri-

marily by phosphorylating the IP3 receptorassociated

cGMP kinase substrate IRAG,229a protein that associates

with PKG and IP3receptor type I to inhibit IP3-induced

calcium release from intracellular stores.230,231Both PKA

and PKG interfere with platelet activation by phosphory-

lating and inactivating VASP (vasodilator-stimulated

phosphoprotein),232a molecule with anticapping activity

that is important for the processes of actin polymeriza-

tion and filopod formation.233-237 PKC can also bind

VASP and interfere with its ability to promote filopodiaformation, although this pathway is unique to collagen-

stimulated platelets and does not involve regulation of

PKA- or PKG-mediated VASP phosphorylation.238

One of the better characterized inhibitory receptors

in platelets is platelet endothelial cell adhesion molecule-

1 (PECAM-1)a cell surface molecule composed of

six extracellular immunoglobulin domains, the most

amino-terminal of which engages in homophilic interac-

tions with PECAM-1 molecules on other cells, and two

cytoplasmic immunoreceptor tyrosinebased inhibitory

motifs (ITIMs) that upon phosphorylation, recruit and

activate the cytosolic SH2 domaincontaining protein

tyrosine phosphatase-2 (SHP-2).239,240

PECAM-1 hasbeen shown to negatively regulate both GPVI- and GPIb/

V/IX-mediated platelet activation241-243perhaps by con-

trolling the phosphorylation state of these two ITAM-

bearing signaling receptorsand appears to be one of

several inhibitory receptors that control the rate and

extent of platelet thrombus formation in vivo.244Platelets

have also recently been found to express two other immu-

noglobulin/ITIM-containing molecules: triggering recep-

tor expressed on myeloid (TREM) cellslike transcript-1

(TLT-1) and products of the G6b gene. TLT-1 is con-

tained within platelet alpha granules and is expressed

on the platelet surface in an activation-dependent

manner.146,245

Although the two cytoplasmic ITIMs ofTLT-1 are capable of becoming phosphorylated and

recruiting SHP-2,146 the extent to which TLT-1/SHP-2

complexes regulate platelet function is not yet known.

The G6bgene, which is located within the class III region

of the human major histocompatibility complex,246gives

rise to multiple alternatively spliced transcripts (G6b-A

through G6b-G).247Platelets contain at least two (G6b-A

and G6b-B)71,86,248 and possibly four (G6b-A, G6b-B,

G6b-D, and G6b-E)249of these transcripts, and the G6b-

B isoform contains cytoplasmic ITIMs that are capable

of becoming tyrosine-phosphorylated and recruiting

SHP-1 and SHP-2.247In platelets, the G6b-B isoform has

been shown to be tyrosine-phosphorylated in resting andactivated platelets, but to associate with SHP-1 only

upon platelet activation.248 Cross-linking of antibodies

specific for G6bgene products has been shown to inhibit

platelet aggregation in response to multiple stimuli249;

however, whether these effects are due to the inhibitory

function of G6b-B remains to be determined.

Several inhibitory pathways have been identified in

platelets that either regulate or are regulated by PI3Ka

lipid kinase that phosphorylates the 3position of PIP2to generate phosphatidylinositol 3,4,5-triphoshate (PIP3),

thereby creating docking sites on the inner face of the

Ca2+

+DAG

CalDAG-GEF1

PKCPKD

1 Rap1RIAM/Talin

Integrinactivation

complex (IAC)

PLCPLC

Bent stalk

+

+

+++++

EGF4

EGF3

EGF2

EGF1PSI

Hybrid

Fully

exp

osed

ligan

dbi

ndin

g

domain

-TD

Calf2

Calf1

Thigh

Clasped tails

IIb

3

Crypticligandbinding

domain

FIGURE 25-6. Integrin activation. As shown in the schematic at thebottom, calcium and diacylglycerol (DAG), generated as a result of the

combined actions of phospholipase (PLC) and PLC, activate twoproteins: (1) protein kinase C (PKC) and (2) the Rap guanine exchange

factor CalDAG-GEF1. Each of these is able to independently activate

the small guanosine triphosphates (GTPase) Rap1. In its GTP-bound

form, Rap1 binds to and activates one of its effector molecules, Rap1GTP-

interacting adaptor molecule (RIAM), which then binds talin to form

an integrin activation complex (IAC). When the IAC binds specific sites

within the 3cytoplasmic domain, the clasp breaks, thereby destabiliz-ing transmembrane domain helix associations that are thought to main-

tain the integrin in its low-affinity state (shown on theleft

). Breaking

the hinge causes extensive conformational changes in the extracellular

domain and produces a high-affinity, ligand-bindingcompetent integ-

rin (shown on the right). EGF, epidermal growth factor; PSI, plexin-

semiphorin-integrin; -TD, beta terminal domain. (Portions adaptedfrom Wegener KL, Partridge AW, Han J, et al: Structural basis of integrin

activation by talin. Cell 2007;128:171, with permission.)


12/20

1390 HEMOSTASIS

plasma membrane for Pleckstrin homology (PH)

domaincontaining molecules.250The actions of PI3K are

opposed by the lipid phosphatase SHIP1 (SH2 domain

containing inositol 5-phosphatase 1, which hydrolyzesthe 5-phosphate of PIP3).251,252In platelets, SHIP1 hasbeen shown to downregulate PIP3generation after IIb3-mediated outside-in signaling253and thus may interfere

with the feed-forward amplification pathways thatincrease the efficiency with which platelets are recruited

to growing thrombi. Interestingly, the 5-inositol phos-phatase activity of SHIP1 appears to be enhanced, at

least in part, by the actions of the Src family tyrosine

kinase Lyn,254-256 which itself has been shown to limit

platelet aggregation in response to GPVI-specific

stimuli257 and after platelet spreading on immobilized

fibrinogen.253

Akt (also known as protein kinase B) is a PH domain

containing serine/threonine kinase that is a well-charac-

terized effector of PI3K.258,259Akt contributes positively

to platelet activation in multiple ways, one of which

appears to be by inactivating the serine/threonine kinaseglycogen synthase kinase 3 (GSK3). The GSK3 family is

composed of three isoforms (, , 2) that are constitu-tively active in resting cells but become inactivated in

activated cells by Akt-mediated phosphorylation.260Plate-

lets express two isoforms of GSK3 ( and ), both ofwhich become phosphorylated and inactivated after

exposure of the platelet to multiple agonists that activate

PI3K and Akt.261 Whereas initial studies reported that

specific inhibitors of GSK3 activity block rather than

enhance platelet responses to agonist stimulation,261 a

recent report suggest that as in other cells, the isoformof GSK3 acts as a negative regulator of platelet function

both in vitro and in vivo.262

ADDITIONAL ROLES FOR PLATELETS INVASCULAR PHYSIOLOGY: VESSEL REPAIR(ANGIOGENESIS), INFLAMMATION, ANDATHEROTHROMBOSIS

In addition to being essential for primary hemostasis,

activated platelets and their secreted products have the

ability to influence a broad array of pathophysiologic

processes, including leukocyte trafficking and inflamma-

tion, tissue regeneration and angiogenesis, and both the

beginning and end stages of atherosclerosis.Activated platelets that become spread on compo-

nents of the extracellular matrix, or on each other, display

an altered surface phenotypethe most prominent of

which is exposure of several thousand copies of the alpha

granulederived membrane protein P-selectin. P-selectin

is also expressed on cytokine-activated endothelial cells.

Thus, after either a thrombotic or inflammatory event,

P-selectin appears on the luminal face of the vessel wall,

where it serves to recruit monocytes and neutrophils into

the underlying tissue by binding PSGL-1a constitu-

tively expressed counter-receptor for P-selectin that is

present on most leukocytes. In vivo, mice lacking P-selec-

tin exhibit greatly diminished leukocyte rolling, delayed

recruitment into sites of inflammation, and increased

susceptibility to infection.263,264Although endothelial P-

selectin no doubt has a major role in leukocyte capture,

platelet P-selectin probably plays a prominent role in

secondary capture.265,266As in platelets, tethering also

initiates activation of leukocyte integrins, which are thenable to mediate cell spreading and transendothelial migra-

tion. P-selectin/PSGL-1 interactions therefore constitute

an important link between thrombosis and inflamma-

tion.150,267 Other platelet/leukocyte receptor/counter-

receptor pairs have also been shown to facilitate the

inflammatory response, including binding of platelet-

associated fibrinogen to the leukocyte integrin MAC-1268

and platelet JAM-3 binding to MAC-1 on monocytes269

and dendritic cells.270

In addition to forming a platform for leukocyte

recruitment during acute inflammation, platelets also

deliver to the vessel wall proinflammatory chemokines

that are thought to play a role in the development ofatherosclerosis by promoting further chemoattraction of

leukocytes and stimulating proliferation of vessel wall

smooth muscle cells and fibroblasts. Such secreted factors

include the C-X-C chemokine platelet factor 4, macro-

phage inflammatory protein 1a (MIP-1a), the C-C

chemokine RANTES (regulated on activation, T cell

expressed and secreted), CD40 ligand, platelet-derived

growth factor (PDGF), and transforming growth factor

(TGF-).150,267,271,272Activated platelets also synthesizede novo IL-1,273,274 a potent stimulator of endothelialcells and monocytes that upregulates adhesion molecule

expression. Thus, platelets appear to contribute in a

number of ways to the development and progression ofatherosclerotic lesions.

Finally, so that one is not left with the impression

that platelets only exacerbate chronic human disease, it

should be noted that platelets and their secreted products

were shown as early as 1969153 to be able to nurture

the vascular endothelium, and they have recently been

proposed as a source of biologic response modifiers for

a plethora of uses, including organ preservation, gum

restoration after dental procedures, and tissue repair after

surgery.152 Their ability to adhere at sites of vascular

injury and secrete both degradative enzymes and at the

same time growth-promoting factors such as vascular

endothelial growth factor (VEGF), PDGF, fibroblastgrowth factor (FGF), epidermal growth factor (EGF),

and angiopoietin 1 allows them to play a uniquely sup-

portive role in endothelial cell migration and survival

during the process of wound healing and

angiogenesis.151

Acknowledgment

The authors thank Robert I. Handin for valuable insights

gleaned from versions of this chapter that appeared in

earlier editions of this book. Research in the authors


13/20


laboratories is supported by grants from the American

Heart Association and the National Heart, Lung, and

Blood Institute of the National Institutes of Health.

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