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REVIEW ARTICLE
From the Cardiovascular Biology Laboratory, Harvard
School of Public Health, Boston, MA; Cardiology Division, Massachusetts
General Hospital, Boston, MA.
The critical role played by platelets in
hemostasis, thrombosis, vascular remodeling, and healing is related to
their function as exocytotic cells that secrete important effector
molecules at the site of vascular injury. Recent insights into
molecular mechanisms of secretion indicate that platelet granule
secretion is homologous to exocytosis in neurons and other cells but
involves a platelet-selective machinery that is uniquely coupled to
cell activation. This review summarizes these recent insights in the context of the burgeoning literature on mechanisms of exocytosis in
neurons and other cells Physiologic role of exocytosis
Platelet granule development and contents Platelet alpha granules contain polypeptides such as coagulation proteins (eg, fibrinogen, factor V), soluble adhesion molecules (eg, von Willebrand factor, vitronectin), growth factors (eg, platelet-derived growth factor, epidermal growth factor), protease inhibitors (eg, plasminogen activator inhibitor-1, 2-antiplasmin), and membrane adhesion molecules (eg,
P-selectin, IIb 3). Platelet dense
granules contain small molecules (eg, ADP, calcium, magnesium) that are
important for activating cells.3
When platelets originate by segmentation from megakaryocytes, they
contain a complex network of intact membrane structures that includes
the plasmalemma, the granules, the surface-connected canalicular system
(SCCS) and the dense tubular system. The development of platelet
granules requires the activation of a platelet-selective genetic
program because deletion of a transcription factor, NF-E2, blocks
granule formation, as well as platelet release from the megakaryocyte.5 Membranes of the alpha granules contain
proteins found on the plasma membrane (eg,
The dense granules contain small molecules such as serotonin that are
taken up and concentrated through specific transporting and storage
mechanisms.29-31 Although the contents of the alpha and
dense granules differ from each other, recent studies have shown the
presence of the alpha granule protein P-selectin and the plasma
membrane proteins Ultrastructural features of secretion Platelet activation causes dramatic cytoskeletal rearrangements that are important for shape change, adhesion, aggregation, exocytosis, and retraction.2,36,37 When platelet exocytosis occurs, granules fuse with the target plasmalemma or SCCS membranes to secrete their cargo into the extracellular space. Some experts favor a model in which granules individually fuse with the SCCS membrane,38,39 whereas others suggest that platelet secretion involves compound fusion of the granules with each other and then with the plasmalemma membrane.40-43 In bovine platelets, which lack an SCCS, the dense granules anchor in close physical apposition to the plasma membrane (in a manner similar to vesicle docking in neurons, see below). These anchored vesicles then fuse with the plasmalemma in response to increased intracellular Ca ++.44At least 3 systems of cytoskeletal fibers are present in the platelet: the membrane skeleton, microtubules, and microfilaments (long actin filaments). When platelets are activated, the percentage of filamentous actin rapidly increases.36,45-47 The membrane skeleton is associated with the network of cytoplasmic actin filaments, various actin-binding proteins, and surface membrane glycoproteins (reviewed in Fox36 and Hartwig and Kwiatkowski48) and is preserved in Triton X-100 lysates of platelets as a continuous layer at the periphery of the cytoplasmic filaments.47 In resting platelets, circumferential bundles of microtubules play an important role in maintaining the discoid shape of platelets. Microtubule depolymerization leads to the loss of the discoid shape.49 Microtubules also appear to play an important role in platelet secretion because colchicine (a tubulin ligand)50 and monoclonal antibodies to alpha and beta subunits of tubulin51 inhibit platelet secretion (when introduced into permeabilized cells). Microtubule-associated proteins regulate the stability and phosphorylation of the microtubules; these proteins may also play a role in microtubule reorganization.52 The contractile ring and stress fibers of platelets may also be involved in exocytosis and retraction.37 Cytoskeletal reorganization is mediated through actin polymerization and depolymerization regulated by phosphatidylinositol-4,5-bisphospate (PIP2), which binds to actin regulatory proteins such as scinderin and gelsolin.53 Consistent with these observations, recombinant scinderin potentiated Ca++-induced serotonin release in permeabilized platelets, whereas PIP2, or 2 peptides that interfere with scinderin-actin interactions, blocked exocytosis.54 In summary, changes in the platelet cytoskeleton are linked to the process of cell activation and appear to play a critical role in granule movement and exocytosis. Unfortunately the mechanisms through which cytoskeletal alterations moderate platelet secretion remain poorly understood.
There are several recent reviews of platelet secretory
defects.40-42,55,56 Congenital defects in
platelet secretion are uncommon, and, the storage pool diseases (SPDs)
are the best characterized abnormalities. Patients with the SPDs may
have abnormal bleeding times and platelet aggregation
defects.57,58 The SPDs have been categorized into 3 groups by electron microscopy: decreased or abnormal alpha granules
( Patients with the The Mutations in different genetic loci have been identified in patients
with HPS and in strains of animals demonstrating HPS-like storage pool
defects such as the mocha and pale-ear mice.70 A
duplication has been identified in a novel gene (HPS1) in 22 patients of Puerto Rican descent with HPS that was not found in normal
controls of Puerto Rican descent or HPS patients who were not Puerto
Rican.71 The HPS1 gene is predicted to encode a
700 amino acid polypeptide of unknown function.72 Pale-ear
mice have defects in platelet dense granules and melanosomes and carry an insertional mutation in an exon of a 3' coding sequence in the
murine counterpart of the HPS locus.73 The mocha mouse has HPS-like storage pool defects involving platelet dense granules, melanosomes, and lysosomes. In these mice the gene for the Although the relationships between the genetic defects of CHS, HPS, and the molecular pathogenesis of abnormal vesicle trafficking and function are not yet fully defined, these insights suggest that normal development of platelet dense granules is related to lysosomal vesicle trafficking. Defects in vesicle formation and trafficking markedly affect downstream processes such as exocytosis, even if the exocytotic machinery is intact.77 The The The empty sack syndrome is another secretory disorder in which there is a reduced level of stored nucleotides and serotonin, but granule membrane proteins are present in normal numbers as measured by granulophysin content.86 Other patients with defective secretion have been identified with disorders that appear to involve impaired aggregation and cell signaling.87-89
Studies of vesicle trafficking and fusion in yeast, neurons, and neuroendocrine cells have provided a conceptual framework for elucidating regulated exocytosis in platelets. Regulated exocytosis is "triggered" by intracellular signal(s) produced by cell activation or membrane depolarization and is a subset of the vesicle trafficking and fusion processes that move molecules in and out of cells. The triggered secretion of neurotransmitters is intensively studied and
has been the subject of several recent reviews.90-97 There
are also recent reviews of exocytosis in
neuroendocrine98,99 and pancreatic cells.100
It is generally agreed that neuronal exocytosis proceeds through a
sequence of vesicle docking, priming, triggering, and fusion that leads
to release of neurotransmitters into the synaptic cleft (Figure
1). Electron microscopy has demonstrated the docking or close apposition of vesicles to the plasma membrane at
the active zone of neurons.101 Although docking is
necessary for exocytosis, only a subset of the vesicles present can be
released rapidly indicating that additional "priming" or activation
of the docked vesicle is necessary. The release of vesicles is
"triggered" by increases in the intracellular Ca++
concentration.
Recent studies of platelet secretion (see below) have indicated that it
bears important similarities to neuronal exocytosis despite the fact
that these cells have different origins (mesoderm versus ectoderm,
respectively). Figure 2 highlights
important similarities and differences between the 2 processes. Neurons contain small synaptic vesicles (SSV) loaded with neurotransmitters such as
There is a marked difference in the kinetics of exocytosis and signal
transduction between these 2 types of cells. Neurotransmitters are
released from SSV within 200 µs, whereas platelet granule release
typically takes 2 to 5 seconds
SNARE machinery The SNARE (soluble NSF attachment protein receptor) hypothesis has been an influential model of general vesicular fusion.106 The SNARE hypothesis states that the vesicle fusion apparatus contains 3 components: t-SNAREs, v-SNAREs, and a soluble SNAP/NSF component (Figure 4). The t-SNAREs are target receptors such as the syntaxins and SNAP-25 that are found on the target membrane (the plasma membrane in the case of exocytosis). The v-SNAREs are vesicle-associated membrane receptors (VAMP or synaptobrevins). The soluble components consist of NSF (N-ethylmaleimide-sensitive factor) and the soluble NSF-attachment proteins (SNAPs;, ,  isoforms). In
yeast cells, vesicle membrane fusion requires 3 sequential steps of
priming, docking, and fusion. Priming is thought to involve
dissociation of the SNARE complex, "activation" of a t-SNARE, and
adenosine triphosphate (ATP)-mediated release of SNAP catalyzed by
NSF.107,108 Docking, the stable association between vesicles and membranes (or between vacuoles), requires a
low-molecular-weight GTPase or Rab-like molecule.109
Fusion between membranes requires the formation of ternary
core complexes between the t-SNAREs and a v-SNARE, but not
NSF.109 The formation of a ternary complex between the
t-SNAREs (SNAP-25 and syntaxin) and the v-SNARE (VAMP) may drive
membrane fusion by overcoming the energy barrier that normally blocks
it (see below).110 It is important to note that priming
has a different meaning for neuroendocrine cells than it does for
yeast. For neuroendocrine cells priming is a Mg-ATP-dependent step
that follows docking that readies vesicles for rapid exocytosis in
response to Ca++ signals (Figure 1), and probably involves
the SNAREs.97,111,112
In neurons, syntaxin 1, SNAP-25, and VAMP 2 form a stable trimolecular SNARE complex.90-97,106 Syntaxin 1 belongs to a large family (at least 16 members) of type 1 integral membrane proteins that are classically found on the plasmalemma or acceptor membrane.97 SNAP-25 in neurons (or related molecules SNAP-23 and SNAP-29 in other cells) attaches to target membranes (ie, plasmalemma) via palmitoylated residues in the middle half of the molecule, making its NH2 and COOH terminal ends free to interact with syntaxin and VAMP. VAMPs 1 and 2 are also type I membrane proteins found in vesicle membranes. The VAMP family includes at least 8 known members.97 It has been postulated that the combinatorial complexity of the syntaxin-SNAP-25-VAMP system may play a role in directing vesicles to the correct target membranes for fusion, but in vitro experiments have not confirmed this hypothesis.113 Although the SNARE complex is sufficient in some systems for mediating vesicle-membrane mixing, the kinetics and regulation of this process in neurons, platelets, and other cells argues that other molecules are involved.97,114,115 The SNARE proteins may participate in the initial formation of a hemifusion stalk between membranes with subsequent generation of the fusion pore, lipid mixing, and exocytosis requiring other, as yet unidentified, molecular interactions.97 Sec1/Munc 18 Members of the Sec1/Munc 18 gene family regulate interactions between the SNARE proteins. The Sec1/Munc 18 proteins bind to the syntaxins and through this binding prevent formation of the core SNARE complex. At the same time the Sec1/Munc 18 molecules have been shown to be required for exocytosis. Sec 1, one of the genes found necessary for the final phase of exocytosis in yeast,116 has a homologue in worms (unc-18) that is required for normal acetylcholine release at the nerve terminal117 and for neurotransmitter release in flies.118 The murine homologue of Sec1/unc-18 (Munc-18-1) binds to syntaxin 1 in neurons and through this binding prevents formation of the SNARE core complex between syntaxin 1, SNAP-25, and VAMP119 that is required for fusion.Deletion of the Munc 18-1 gene in mice is reported to be lethal because it prevents vesicle exocytosis even though it does not interfere with attachment of vesicles to the plasma membrane.97 The 3-dimensional structure of the neuronal Sec1-syntaxin complex has recently been solved.120 This structure has suggested that the Sec1 proteins may provide specificity for the pairing of vesicles with the correct target membranes in addition to modulating SNARE complex formation. Excitation and secretion coupling molecules Although yeast models are useful for understanding constitutive vesicular secretion, they do not account for the regulated exocytosis "triggered" in specialized secretory cells, such as neurons or neuroendocrine cells, by second messengers such as Ca++ transients (reviewed in Calakos and Scheller91). The Ca++ sensors involved in regulated exocytosis are likely to differ between cell types and secretory processes.98 Potential Ca++ sensor/effector molecules identified in neurons include synaptotagmin 1, Munc-13, and Doc2.121 These proteins contain protein kinase C-C2 or potential Ca++-binding domains. Synaptotagmin 1 knockout mice show impaired fast Ca++-dependent vesicle exocytosis in neurons121,122 arguing that this protein plays a critical role in Ca++-triggered secretion in these cells. Munc13 (the murine homologue of unc13) contains a protein kinase C-C1 or diacylglycerol (DAG)-phorbol ester-binding domain, translocates to the plasma membrane, and enhances phorbol ester-induced release of neurotransmitters at neuromuscular junctions.123,124 Unc-13 is necessary for normal synaptic function in Caenorhabditis elegans.125,126 Munc 13 can displace Sec1 (unc18) from syntaxin and deletion of the Munc13-1 gene in mice causes impaired synaptic vesicle release in response to action potentials.127,128 However, because Munc-13 does not appear to bind Ca++, its role as a direct Ca++ sensor has been challenged.127 The C2 domains of Doc2 interact with Ca++ and phospholipid, which may target it to synaptic vesicles and other membranes.129 One domain of Doc2 binds the murine Sec1 protein (Munc18) and competes with Munc18-syntaxin binding, whereas another domain of Doc2 interacts with Munc13 in a manner that is enhanced by Ca++ and phorbol ester.130,131 In genetic experiments, overexpression of Doc2 increases secretion in PC12 cells, suggesting that this protein plays a critical role in exocytosis.132 Several other potential Ca++ sensors have been proposed including the annexins, calcyclin, calmodulin, unc31, frequenin, rim, and scinderin.133 Although these molecules may prove to modulate other Ca++-dependent steps in the exocytotic pathway, the late-acting or triggering Ca++ sensor is likely to interact directly with the SNARE proteins because of their critical role in the initiation of vesicle fusion.Other modulators of exocytosis Abundant evidence implicates the Rab proteins, a large family of Ras-related low-molecular-weight GTPases in vesicle trafficking and fusion. At present the precise role played by the Rab proteins in exocytosis remains unclear despite evidence that these molecules interact with the SNAREs in yeast 134-136 and are involved in vesicle docking, in a step between NSF/SNAP action and fusion.107 The Rab proteins may exert their effects through effector molecules that specifically interact with the guanosine triphosphate (GTP)- or guanosine diphosphate (GDP)-bound form of the molecule. Deletion of the rab3a gene in mice causes a mild phenotype with an increase in the number of vesicle fusion events elicited by an action potential137; this suggests that Rab3A may play an inhibitory role in neuronal exocytosis.
Activation-secretion coupling The platelet is activated (Figure 3) when specific physiologic ligands (eg, ADP, thrombin, thromboxane A2, platelet activating factor, collagen, epinephrine) interact with cognate receptors on the plasma membrane. Receptor engagement activates phospholipase C either via G-protein-coupled mechanisms (eg, thrombin, reviewed in Brass et al138) or via other signaling mechanisms (eg, collagen reviewed in Barnes et al139). Phospholipase C cleaves PIP2 to inositol-1,4,5-trisphosphate (IP3) and DAG. In turn IP3 increases cytosolic Ca++ from 40 to 100 nmol/L to 2 to 10 µmol/L, which initiates granule secretion.140-145 There is strong evidence in platelets that protein kinase C also plays an important role in inducing secretion. Platelets contains several isoforms of protein kinase C (,I,II, , , , ), many of
which are activated by DAG and Ca++.146 After
cellular activation by thrombin, protein kinase C phosphorylates
pleckstrin, myristoylated-alanine rich C kinase substrate (MARCKS), and
several other substrates within seconds, with kinetics that parallel
the kinetics of platelet secretion and aggregation.147-149
Phorbol esters mimic DAG in their stimulation of protein kinase C; they
induce platelet secretion and aggregation and potentiate the effects of
calcium ionophores or agents that increase
Cai++.150-152 Inhibitors of protein kinase
C block dense granule secretion153 and have been used to
demonstrate synergism between Cai++ and protein kinase
C activity in platelet secretion.154 Although the protein
kinase C isoform(s) required for platelet secretion is not known,
protein kinase C has been shown recently to be necessary for
exocytosis in mast cells.155
A potential role for low-molecular-weight GTP-binding proteins (eg, the
Rabs and other GTPases) in exocytosis has been suggested because the
exocytotic effects of Ca++ are potentiated by GTP- Current knowledge Recent studies have revealed that the secretory machinery in platelets has important homologies to the machinery found in neurons and other cells. Platelets can form core SNARE complexes in vitro that support SNAP-dependent NSF-ATPase activity158 and thisSNAP-dependent NSF ATPase activity has been shown to be critical for
exocytosis of both alpha and dense granules.159 Platelet
membranes contain syntaxins 2 and 4, which play distinctive roles in
granule exocytosis; syntaxin 2 is involved in dense granule release,
whereas syntaxin 4 is necessary for platelet alpha granule secretion.158,160-162 Platelets contain abundant amounts
of SNAP-23, and a VAMP, which interact and form SNARE complexes that
reportedly dissociate after cell activation (Figure
4).160,161 SNAP-23 is required for dense granule
exocytosis, but it is not yet clear which VAMP is functionally involved
in secretion.162,163 A platelet Sec1 protein (PSP) has
been cloned, which is orthologous to Munc-18c.161 PSP
forms a tight complex with syntaxin 2 and syntaxin 4 that can prevent
the formation of the SNARE complex (Figure 4)161,164; antibodies against PSP inhibit dense granule secretion in permeabilized cells (J. Polgar and G. L. Reed, 1998, unpublished observations). PSP
is phosphorylated in platelets activated by thrombin through a protein
kinase C-dependent mechanism. Protein kinase C-phosphorylation of PSP
inhibits syntaxin 4 binding, which relieves the inhibitory effect of
this molecule on SNARE complex formation.161
Alternatively, other as yet uncharacterized platelet molecules, such as
homologues of Doc2, Munc 13, and so on, may induce a conformational
change that releases PSP from syntaxin (Figure 4).120
Platelets also contain a number of different Rab molecules including Rab 3b, 6, 8, 11, 31.77,165 Rabs 3b, 6, and 8 are phosphorylated when cells are activated by thrombin. Rab 6 phosphorylation proceeds through protein kinase C mechanism and is associated with alterations in the GTP/GDP binding affinities and cell localization.165,166 To date, no direct functional role has been established for the Rabs in platelet secretion.
At present very little information is available about the mechanisms of secretion in leukocytes or endothelial cells. It has been argued that leukocytes use lysosomes for both storage and exocytosis, unlike typical secretory cells, which use separate organelles for storage and release of their secretory products.167 SNARE proteins have been found in neutrophils, and phagocytosis appears to occur through a SNARE-dependent mechanism.168-172 In mast cells, SNAP-23 relocation from the plasmalemma to the granule membrane is required for compound granule-granule, granule-plasmalemma fusion typical of these cells.173 Overexpression of Rab 3d in rat basophilic leukemic cells (RBL-2H3 cells) inhibits exocytosis of hexosaminidase stimulated by interactions with the high-affinity IgE receptors.174 Evidence indicates that endothelial cell secretion also occurs through an NSF-SNARE-dependent mechanism175 though NSF-dependence has not been found by other investigators.176
Despite their unique characteristics, platelets use a molecular machinery for exocytosis that is homologous to that used in other types of secretory cells (eg, neurons) and different organisms (yeast to humans). However, many of the important physiologic differences between platelets and neurons in the regulation and kinetics of exocytosis are due to the fact that platelet secretory molecules arise from different genes and have distinctive features. The core SNARE-related molecules responsible for platelet secretion have recently been identified. Now the regulatory linkages or signaling mechanisms that link these molecules and the process of platelet activation need to be elucidated more completely. Because the coupling of cell activation to exocytosis differs significantly between platelets and neurons, it is likely that the molecules that transduce these processes in platelets will be quite different from their counterparts in neurons. Unlike neuronal exocytosis, secretion in platelets is triggered by extracellular ligand-receptor interactions that lead to cell activation. These receptors are coupled to second messengers, such as Ca++, DAG, and protein kinase C, that signal to the exocytotic machinery. Recent studies have demonstrated that thrombin activation of platelets induces protein kinase C-mediated phosphorylation of PSP, providing important linkages between thrombin activation and the secretory machinery.161 Increased Cai++ is a critical determinant of secretion in platelets but the molecular sensor(s) in platelets that transduces or couples the Cai++ signal to secretion must still be discovered. Platelets represent a unique opportunity for examining exocytosis in a cellular system that is not confounded by the membrane trafficking events found in actively synthetic cells. Beyond studies of the exocytotic machinery, important insights into secretion have come from physiologic, pharmacologic, ultrastructural, and clinical investigations. It is clear from ultrastructural analyses and from studies of patients with secretory deficiencies, that platelet exocytosis is the net result of several molecular and cellular processes acting in sequence. These processes include the formation, packaging, "differentiation," and maintenance of secretory granules. The molecular mechanisms underlying these processes are important areas for future research. Valuable insights into this process are likely to come from identification of the genes responsible for secretory pool disorders in humans and animals. A particularly striking difference between platelets and nonhematopoietic cells is the strong functional association between changes in platelet shape (mediated by the cytoskeleton) and the exocytotic process. Further studies are necessary to define these linkages at the molecular level. Although many of the signaling pathways involved in platelet activation have been identified, the linkages of these signals to the secretory machinery are poorly understood, even though patients with impaired platelet activation-secretion coupling mechanisms have been described. The fact that some of the patients with platelet storage pool disorders do not appear to have functional defects in their other secretory cells suggests that there may be a megakaryocyte- and platelet-selective molecular machinery for granule formation, packaging, maintenance, and exocytosis that is responsible for the special function of the alpha and dense granules. Insights into the molecular mechanisms responsible for platelet
exocytosis will help define the causes of human platelet secretory disorders. These discoveries will provide general clues for
understanding exocytosis in other vascular cells. Insights into these
mechanisms may also be useful for designing a new class of therapeutic
molecules to reduce the role played by platelet secretion in thrombosis and vascular remodeling
The authors are grateful to the other members of the laboratory, particularly Sul-Hee Chung, Aiilyan Houng, and Lin Liu. Given the scope of this review, it has not been possible to discuss many important contributions to the field of platelet secretion, and the authors apologize to researchers whose work has not been cited.
Submitted February 11, 2000; accepted May 23, 2000.
Supported in part by National Institutes of Health grant HL-64057.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Guy L. Reed, Cardiovascular Biology Laboratory, Harvard School of Public Health, II-127, 677 Huntington Ave, Boston, MA 02115; e-mail: reed{at}cvlab.harvard.edu.
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Top
Introduction
Overview of platelet secretion
illustrating the fundamental homologies and
the unique characteristics of platelet secretion.
