Platelets and the Vessel Wall

Platelet Morphology and Subcellular Organization

Platelets are adhesion and signaling machines that cir­culate as small, disc-shaped cellular fragments in the whole blood of healthy individuals at a concentration of approximately 150,000 to 300,000 per µL. Early studies suggested that platelets might be produced via cytoplasmic fragmentation along a network of internal demarcation membranes that were observed in large, polyploid megakaryocytes. More recent studies, however, sup­port the notion that proplatelets are assembled and packaged with their various constituents at the ends of long cytoplasmic extensions of differentiated megakaryocytes that have migrated from the proliferative osteoblastic niche to the capillary-rich vascular niche of the bone marrow microenvironment, with the invaginated demarcation membrane system serving simply as a reservoir of internal membrane used for proplatelet extension. Once adjacent to the adluminal face of the endothelium, proplatelets are released into the blood stream, where they circulate as mature platelets for approximately 7 to 10 days before being cleared by the liver and spleen, their life span being controlled at least in part by an antagonistic balance between the apoptotic proteins Bcl-xL and Bak.

The size of resting platelets is somewhat variable, averaging approximately 1.5 µm in diameter and 0.5 to 1 µm in thickness. Platelet size is undoubtedly regulated by numerous factors during their biogenesis, but both the 224-kDa nonmuscle myosin heavy chain IIA (MYHIIA) and the cell-surface glycoprotein Ib complex (GPIb) appear to play critical roles. Thus mutations in the MYH9 gene act dominantly to interfere with contractile events important for platelet formation, whereas failure to express GPIb results in Bernard-Soulier syndrome by disruption of critical associations with the cytoskeletal protein filamin, which play an essential role both in platelet formation and in platelet compliance. In both of these inherited platelet disorders, platelets are as large as lymphocytes (see Chapter 30 ). Correction of GPIb expression in GP Ib-deficient (Bernard-Soulier syndrome) mice has been shown to restore platelets to their normal size.

The volume of platelets (mean platelet volume) normally ranges from 6 to 10 femtoliters. Platelet density is also variable, and the issue of whether young platelets are more or less dense as they gain versus lose content during their circulating lifetime has never been satisfactorily resolved. Because platelets retain most species of messenger ribonucleic acid (mRNA) for a short period after their release from bone marrow megakaryocytes, young platelets can be distinguished from older ones based on their RNA content.

As shown in Figure 26-1, A and C , resting platelets are discoid in shape, largely because of the presence of a circumferential coil of microtubules, and are packed with numerous electron-opaque α granules, a few dense (δ) granules, several mitochondria, and lysosomes. Platelets also retain a few Golgi bodies, as well as occasional vestiges of rough endoplasmic reticulum—the exception being platelets from patients with rapid platelet turnover, where very young platelets containing more abundant protein synthesis machinery are readily observed in the circulation. Platelets also contain two highly specialized membrane systems not found in other cells of the body: the surface-connected open canalicular system (OCS; see Fig. 26-1, B and C ), and the dense tubular system (DTS). The OCS is a series of tortuous invaginations of the plasma membrane that appear to tunnel throughout the cytoplasm of the cell that serves as an internal reservoir of plasma membrane that is called upon when platelets round up, extend lamellipods and filopods (see Fig. 26-1, B and D ), and spread during platelet activation—a process that can increase by more than 400% the surface area of exposed plasma membrane. Because OCS channels are proximal to internal granules, they also likely function as a conduit for the rapid expulsion of α- and δ-granule contents during platelet activation. The DTS, on the other hand, is a remnant of the smooth endoplasmic reticulum and 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 and is thought to contain a 100-kDa calcium adenosinetriphosphatase (ATPase) known as sarcoplasmic/endoplasmic reticulum Ca ²⁺ -ATPase 2b ( SERCA2-b) that 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 DTS, whereas activation of the GPIb/V/IX receptor for VWF releases calcium primarily from a poorly defined acidic compartment within the cell. Thrombin, a strong platelet agonist, appears to elicit calcium release from both stores upon binding to the platelet thrombin receptor PAR-1.

The platelet cytoskeleton is comprised of a single, rigid but dynamic microtubule of approximately 100 µm in length coiled 8 to 12 times around the equatorial plane of the cell. This 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 colchicine, an agent that dissolves microtubules, results in their rounding ; 2) platelets from mice lacking β ₁ tubulin remain largely spherical ; and 3) “platelet spherocytosis” in humans results when tubulin fails to polymerize normally into microtubules. Directly underneath the plasma membrane lies an intricate, two-dimensional, tightly-woven membrane skeleton comprised of nonerythroid spectrin, a network of actin filaments, vinculin, and the actin-binding protein filamin, which itself is tethered to the inner face of the plasma membrane via linkages with the cytoplasmic domain of GPIb. The membrane skeleton, because of its location, serves as a scaffold, linking elements of the plasma membrane with contractile elements of the cytoskeleton and cytosolic signaling proteins, and thereby regulates such diverse functions as receptor mobility, receptor clustering, and signal transduction. Finally, the platelet is filled with an extensive cytoplasmic network of actin filaments organized by the actin-binding proteins filamin and α-actinin that constitute its cytoskeleton.

When platelets become exposed to components of the extracellular matrix or to soluble agonists like ADP or thrombin, they undergo dramatic changes in their morphology. The marginal band of microtubules disappears, allowing the platelet to transform from a disc to an irregular sphere. At nearly the same time, the actin filament capping protein, α-adducin, becomes phosphorylated and dissociates from existing F-actin filaments, exposing the barbed end of the filament to cytosolic actin monomers and driving rapid polymerization of actin into microfilaments. This has the dual effect of driving the extension of lamellipodia and filopodia, and of forcing granules towards the center of the platelet where they can fuse with membranes of the OCS and release their contents to the exterior of the cell. Phosphorylation of myosin additionally induces contractile events that facilitate centralization of the granules.

Platelet Genomics and Proteomics

Though anucleate, platelets contain measurable and manipulable levels of megakaryocyte-derived mRNA, at least some of which is capable of being synthesized into small but detectable amounts of protein. Both serial analysis of gene expression (SAGE) and gene microarray analyses were employed early on to estimate the size and composition of the platelet transcriptome. Of the 20,563 genes present in the human genome, 9538 distinct transcripts (6493 in the mouse) were identified in unstimulated platelets using RNA sequencing (RNA-SEQ). That is considerably fewer than normally found in a nucleated cell, but perhaps more than might have been expected from an anucleate circulating cellular fragment. One of the more surprising findings in recent years has been the identification of heavy (sometimes called 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 be thereafter translated into protein. Enlisted during the activation process, signal-dependent protein translation has thus far been demonstrated for mRNAs encoding interleukin (IL) 1β, tissue factor, and Bcl-3, the protein products of which have the potential to influence inflammation, thrombosis, and wound repair. Platelets also contain an abundance of microRNAs (miRs) that have the potential, especially in precursor megakaryocytes, to regulate transcription and translation.

The platelet proteome appears to be equally complex and diverse, and unlike the transcriptome, reports both the breadth as well as the relative amounts of protein products actually present in the cell. Obtained by refined two-dimensional gel electrophoretic techniques that were originally developed in the 1970s or by liquid chromatographic separation, proteins are fragmented and separated using a combination of proteolytic and ionization techniques and then analyzed by mass spectrometry, allowing for the identification of thousands of proteins present in complex cellular lysates or subcellular fractions. In by far the most complete and quantitative pro­filing analyses to date, Burkhart and colleagues have identified a core platelet proteome comprised of approximately 5000 distinct platelet proteins, including an abundance of molecules involved in adhesion, signaling, cytoskeletal change, and metabolism. Others have combined prefractionation methods with suitable separation techniques to describe the inventory of platelet proteins that become phosphorylated during platelet activation, can be palmitoylated, or are enriched within various subcellular compartments such as the platelet cytoskeleton, platelet α-granules, the membrane fraction, membrane rafts, and microparticles.

Antithrombotic Components of the Vessel Wall

Though hundreds of thousands of platelets per microliter circulate in blood, very few, if any, interact with the intact vessel wall, because the endothelial lining of the blood vessel presents an excellent nonthrombogenic surface. In fact, this property of the vessel wall has not yet been duplicated in any prosthetic or extracorporeal device. The healthy endothelium not only provides an effective barrier between blood and the highly thrombogenic components of the subendothelium (see below), but also actively produces both membrane-bound and secretory products that limit fibrin generation and promote clot dissolution. For example, heparinlike glycosaminoglycans present on the luminal side of the endothelial cell surface recruit plasma antithrombin, effecting a conformational change that promotes binding and neutralization of thrombin and other serine proteases. Thrombin, when bound to the endothelial 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, suppressing further thrombin generation. Endothelial cells also express a specific receptor for activated protein C, which serves to concentrate the protein on the endothelial surface. Finally, endothelial cells synthesize, secrete and rebind tissue plasminogen activator, which activates plasminogen to facilitate fibrin dissolution ( Fig. 26-2 ).

The endothelial cell also produces two important inhibitors of platelet activation: prostacyclin (PGI ₂ ) and nitric oxide (NO). The latter was first recognized for its role as an endothelium-derived relaxation factor. A labile oxygenated metabolite of arachidonic acid generated by endothelial cell cyclooxygenase-2 (COX-2), PGI ₂ diffuses out of the cell and binds to a platelet G _s -protein coupled receptor (GPCR) known as the isoprostenoid (IP) receptor. Its release stimulates adenylate cyclase to increase cytosolic cyclic adenosine monophosphate (cAMP) levels, which 1) activates a pump in the DTS that decreases cytosolic calcium ions (Ca ²⁺ ), thus helping keep platelets quiescent, and 2) activates protein kinase A (PKA). PGI ₂ also has potent vasodilatory effects, binding to IP on arterial smooth muscles cells to effect vessel relaxation. PGI ₂ produced by the vascular endothelium thus serves to counterbalance the proaggregatory and vasoconstrictor activities of the platelet-derived prostanoid, TXA ₂ , the biology of which is discussed below. In fact, upsetting the delicate balance between COX-1–derived TXA ₂ and COX-2–derived PGI ₂ has been shown to increase the risk of adverse cardiovascular events.

Whereas PGI ₂ stimulates adenylate cyclase to produce cAMP, NO, a product of L-arginine generated by endothelial nitric oxide synthase (eNOS), directly activates platelet guanylate cyclase, resulting in increased levels of cytosolic cyclic guanosine monophosphate (cGMP). Although platelet responses to low levels of this cyclic nucleotide can at first be mildly stimulatory, cGMP, largely via its activation of protein kinase G (PKG), has the overall effect of dampening platelet responses and inhibiting platelet adhesion, platelet aggregation, and platelet-mediated recruitment of leukocytes during the inflammatory response (see Fig. 26-2 ). In addition to these two soluble metabolites, endothelial cells also express on their surface a potent ADPase known at CD39, which scavenges plasma ADP to prevent platelet aggregation. Finally, it is important to note that inflammatory cytokines, oxidized lipids, and immune complexes can, under pathologic conditions, inhibit these protective biochemical pathways and impair the antithrombotic state of the endothelial cell. These changes permit untoward formation of platelet- and fibrin-containing thrombi, as well as thrombus formation beyond sites of vascular injury, and can thus contribute to atherothrombosis.