Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro-and antiangiogenic proteins are separated in distinct subpopulations of ␣-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct ␣-granules. These observations motivated the hypothesis that distinct populations of ␣-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH 2 ) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH 2 ) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of ␣-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis. IntroductionAngiogenesis, the process of new vessel development, plays an essential role in embryogenesis, but postnatal angiogenesis is limited to sites of abnormal vascular surface. An activated vascular endothelium can be induced by tissue injury or wound healing, by hormonal cycling such as in pregnancy and ovulation, or by tumor-induced vessel growth. In all of these circumstances, platelets act as the initial responder to vascular change and provide a flexible delivery system for angiogenesis-related molecules. [1][2][3][4] The process of postnatal angiogenesis is regulated by a continuous interplay of stimulators and inhibitors of angiogenesis, and their imbalance contributes to numerous inflammatory, malignant, ischemic, and immune disorders. 5 There is a revived interest in the overlap between angiogenesis and platelets 6 because several clinical trials have now shown that anticoagulation can improve cancer survival 7,8 beyond the benefit derived from the treatment of deep vein thrombosis alone.It is known that platelets stimulate endothelial cells in culture and can promote the assembly of capillary-like structures in vitro. 9,10 Platelets may modulate angiogenesis by releasing promoters such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and matrix metalloproteinases (MMPs). 1,6,[11][12][13][14][15][16][17][18] The repertoire o...

New steps in the process of conversion of proplatelet extensions from megakaryocytes into mature platelets are defined.

Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocytederived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P ؊ , LAMP-1 ؊ and expressed fulllength filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P ؊ and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet micropar-

The vasculature is a highly specialized organ that functions in a number of key physiological tasks including the transport of oxygen and nutrients to tissues. Formation of the vascular system is an essential and rate-limiting step in development and occurs primarily through two main mechanisms, vasculogenesis and angiogenesis. Both vasculogenesis, the de novo formation of vessels, and angiogenesis, the growth of new vessels from pre-existing vessels by sprouting, are complex processes that are mediated by the precise coordination of multiple cell types to form and remodel the vascular system. A host of signaling molecules and their interaction with specific receptors are central to activating and modulating vessel formation. This review article summarizes the current state of research involving signaling molecules that have been demonstrated to function in the regulation of vasculogenesis and angiogenesis, as well as molecules known to play a role in vessel maturation, hypoxia-driven angiogenesis and arterial-venous specification.

The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTPrhodamine-tubulin revealed tubulin incorporation at 7.9 (؎ 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (؎ 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP-expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states. IntroductionPlatelets are cells that function in maintaining vascular integrity. Their disclike shape enables platelets to travel along the apical endothelium of vessels, where they respond to vascular damage by activating and releasing hemostatic factors. Situated beneath the plasma membrane is a circumferential marginal band composed of 7 to 12 filamentous rings that maintain the discoid shape of resting platelets. Marginal bands assemble in blood cells of other species, but platelets are the only mature human cell possessing a circumferential marginal band. 1 The platelet marginal band is composed almost entirely of microtubules. 2,3 Microtubules are polymers of ␣␤-tubulin dimers that first associate into linear arrays called protofilaments. Protofilaments laterally associate forming the hollow rigid tubular structure characteristic of microtubules. ␤1-Tubulin, a divergent ␤-tubulin isoform exclusive to megakaryocytes and platelets, 4,5 makes up the bulk of ␤-tubulin within the microtubule coil. 3,6 The marginal band preserves the elliptic shape of resting platelets. Transgenic mice lacking ␤1-tubulin possess nondiscoid platelets with defective marginal bands containing only 2 to 3 microtubule coils. 7,8 ␤1-Tubulin-deficient mice experience thrombocytopenia (platelet counts Ͻ 50% of wild-type) and prolonged bleeding times. 8 Chilling of platelets from wild-type mice disassembles their microtubules and induces spherocytosis (spherical shape). Stabilization of the microtubule coil with paclitaxel prior to chilling prevents spherocytosis ...

Summary. The cellular and molecular basis of the intricate process by which megakaryocytes (MKs) form and release platelets remains poorly understood. Work has shown that proplatelets, long cytoplasmic extensions made by mature MKs, are essential intermediates in platelet biogenesis. Microtubules are the main structural component of proplatelets and it is microtubule sliding, driven by dynein motors within cortical bundles, which elongates and thins proplatelets. Kinesin motors carry their cargo of platelet-specific granules and organelles into the proplatelets using the microtubule bundles as tracks. Extension of proplatelets is associated with repeated actindependent bending and bifurcation, which results in considerable amplification of free proplatelet ends. Large proplatelets, dissociated from the residual MK cell body, have the capacity to mature platelets. Only the ends of proplatelets form marginal microtubule coils similar to that observed in mature platelets, demonstrating that platelet formation completes primarily at proplatelet ends. Understanding the molecular basis of platelet formation requires detailed knowledge of how the MK microtubule machinery interacts to generate proplatelets and release platelets.

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver proand antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/ endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This plateletglycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions. (Blood. 2012;120(3):626-635) IntroductionGlycosylation of proteins and lipids has a wide range of biologic functions. 1 The glycosylation apparatus of nucleated cells is primarily located in the secretory pathway throughout the endoplasmic reticulum-Golgi stacks and consists of more than 200 glycosyltransferases, most of which are type II transmembrane-anchored proteins with distinct localization. Their topology within these compartments is directed by signal motifs contained in their stem, transmembrane, and cytoplasmic domains. 2,3 Golgi-located membrane-bound glycosyltransferases are believed to be retained in the appropriate compartments by coat protein I-mediated retrograde transport 3 and to be released and secreted only after proteolytic cleavage in their stalk regions to yield soluble, catalytically active truncated enzymes lacking their N-terminal transmembrane segment. 4 Several previous studies have described unusual subcellular localization of glycosyltransferases outside of their normal confinement to the endoplasmic reticulum-Golgi (referred to herein as ectopic localization). Methodologic ambiguities, however, have cast doubt on these findings. 5 One notable exception concerns reports of glycosyltransferase activities associated with blood platelets. [6][7][8][9][10][11] Platelets are anucleated megakaryocyte subfragments that participate in hemostatic, inflammatory, and host defense reactions. 12 Moreover, platelets deliver pro-and antiangiogenic factors throughout the vascular system. 12 Glycosyltransferase activities in platelets were described 30 years ago and are propo...

Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer-disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrindisrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the "barbell shapes" of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure. (Blood. 2011;118(6): 1641-1652) IntroductionBlood platelets, like erythrocytes, must withstand high shear forces during circulation. Retaining their discoid shape is critical to platelets, because their small size and shape cause them to be propelled by blood flow to the endothelial surface, where they are positioned to readily sense and respond to vascular damage. To provide structural support and prevent gross deformations as they circulate, mature platelets contain a robust membrane skeleton that is formed by spectrin molecules, adducin, and actin filament barbed ends. [1][2][3] Two thousand spectrin tetramers, 200-nm-long head-tohead assemblies of ␣␤ heterodimers, compose the bulk of this 2D network. Although less is known about how the spectrin-actin network forms and connects to the plasma membrane in platelets relative to erythrocytes, certain differences between the 2 membrane skeletons have been recognized. First, spectrin strands comprising the platelet membrane skeleton interconnect on the ends of long actin filaments originating from the cytoplasm instead of short actin oligomers. [3][4][5] Therefore, the platelet spectrin lattice and its associated actin filaments assemble into a continuous ultrastructure. Second, tropomodulins do not appear to have a major role in capping actin filament pointed ends, as occurs in erythrocytes. 6,7 Instead, a substantial number of these ends exist free or are capped by Arp2/3 in the res...