Expression of junctional proteins in human platelets

Platelets play a major role in thrombosis and hemostasis by binding the sub-endothelial matrix at sites of injury, but also participate in vascular pathologies such as atherosclerosis. Recently, junctional proteins like PECAM-I and JAM-family members have been recovered from platelets, therefore we examined what other junctional molecules may be present in platelets. We observed immunoreactivity for APC (147 kD), beta-catenin (92 kD), E-cadherin (120 and 84 kD) and occludin (70-85 kD) by western blotting. Additionally, beta-catenin, pan-reactive cadherins, E-cadherin and occludin were seen to partition with the triton insoluble cytoskeleton in platelets. These proteins were also found in a megakaryocyte (platelet precursor) line, MEG-01. Our data suggest that conventional junctional molecules are expressed in platelets and could possibly participate in aggregation, clot formation and wound healing.     

Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis

An association between platelets, angiogenesis, and cancer has long been recognized, but the mechanisms linking them remains unclear. Platelets regulate new blood vessel growth through numerous stimulators and inhibitors of angiogenesis by several pathways, including differential exocytosis of angiogenesis regulators. Herein, we investigated the differential release of angiogenesis stimulators and inhibitors from platelets. Activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF, but not endostatin whereas, thromboxane A(2) (TXA(2)) released endostatin but not VEGF. Platelet releasates generated by activation with ADP promoted migration and formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in in vitro angiogenesis models. Conversely, TXA(2)-stimulated platelet releasate inhibited migration and formation of capillary structures. Because tumor growth beyond 1-2 mm(3) is angiogenesis-dependent, we hypothesized that cancer cells preferentially stimulate platelets to secrete their pro-angiogenic payload. In support of this, the breast cancer cell line MCF-7 stimulated secretion of VEGF and a pro-angiogenic releasate from platelets. Furthermore, the antiplatelet agent aspirin inhibited platelet-mediated angiogenesis after exposure to ADP or MCF-7 cells providing a potential mechanism for how aspirin may impact malignancy. Manipulation of differentially mediated release of angiogenic factors from platelets may provide a new modality for cancer treatment.     

Lipoxygenase product controls the regulatory volume decrease of human platelets

SUMMARY. Blood platelets exposed to hypotonic medium are known to undergo a regulatory volume decrease (RVD), mediated by increased conductive permeability of K(+) and Cl(-). It is presently shown that RVD in platelets is controlled by a lipoxygenase product, apparently by a selective regulation of K(+) permeability. This conclusion is supported by the following observations: (a) lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), N-(3-phenoxycinnamyl)-acetohydroxamic acid (BW A4C), methyl 2-[(3,4-dihydro-3,4-dioxo-naphthalenyl)amino]-benzoate (CGS 8515), and 5,8,11,14-eicosatetraynoic acid, inhibit RVD with IC(50) values of 3,6,10, and 5 μM, respectively. In contrast, aspirin and indomethacin, known cycloxygenase inhibitors, are innocuous; (b) an eluate from platelets undergoing RVD restores RVD in NDGA-treated platelets; (c) the eluate is unstable (t(1/2) = 8 s and 2 min in the presence and absence of platelets, respectively); furthermore, albumin promotes platelet RVD; (d) known lipoxygenase products, 12-hydroperoxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid and leukotriene D(4), restore RVD in NDGA-treated platelets at 0.1-1.0 μM; (e) the extended hypotonic swelling of gramicidin-treated platelets, expressing Cl(-) permeability, is insensitive to NDGA. It is hypothesized that the lipoxygenase product selectively opens K(+) channels in platelets, in analogy with the effect of lipoxygenase products in cardiac atrial cells and Aplysia sensory neurons.     

On the preservation of contractile proteins during storage of human platelets

A study has been undertaken to determine the rate at which stored platelets lose their ability to respond to stimuli and to establish whether this decrease in function could be ascribed to the storage- induced proteolysis of prominent platelet proteins observed by others. Platelet concentrates were stored at 4 degrees C and 25 degrees C for up to 14 days, and their ability to secrete and aggregate in response to appropriate stimuli was determined at 6, 96, and 192 hr after venipuncture. At each time point the protein complement of the platelets was also monitored by SDS-polyacrylamide gel electrophoresis to assess the extent of intracellular protein degradation. Platelets from concentrates stored at either temperature exhibited a decreased ability to respond to stimuli as storage time increased. After 8 days of storage at 4 degrees C and up to 9 days at 25 degrees C, no proteolysis of major platelet proteins was observed; however, complete loss of platelet function was observed. This strongly indicates that a decrease in platelet function should not be causally linked to degraded contractile-structural proteins and that extending the functional life of platelets during storage is still an attainable goal since proteolysis is not the inevitable result of short-term storage.     

Normal reference ranges for aortic diameters in preterm infants

Objective

To establish normal reference ranges and Z-scores for aortic diameters in preterm infants according to the body surface area and assess their correlation with body weight, body surface area, and gestational age.

G proteins and low-molecular-weight GTP-binding proteins in platelets

Platelets contain at least two distinct families of proteins that can bind GTP: the G proteins and the low-molecular-weight GTP-binding proteins. G proteins are heterotrimeric regulatory proteins that mediate the interaction between cell surface receptors and the enzymes that generate second messengers during platelet activation. At least five different G proteins have been identified in platelets. The low-molecular-weight GTP-binding proteins range in size from 21 to 28 kDa. Little is known for certain about their function, but at least 15 such proteins have been identified in platelets, many of which have been shown to be homologous to the products of the ras protooncogene. In cells other than platelets, low-molecular-weight GTP-binding proteins have been implicated in protein transport, cell activation events, and malignant transformation. Their role in platelets is unknown.     

The functional role of platelets in the regulation of angiogenesis

Functionally, platelets are primarily recognized as key regulators of thrombosis and hemostasis. Upon vessel injury, the typically quiescent platelet interacts with subendothelial matrix to regulate platelet adhesion, activation and aggregation, with subsequent induction of the coagulation cascade forming a thrombus. Recently, however, newly described roles for platelets in the regulation of angiogenesis have emerged. Platelets possess an armory of pro- and anti-angiogenic proteins, which are actively sequestered and highly organized in α-granule populations. Platelet activation facilitates their release, eliciting potent angiogenic responses through mechanisms that appear to be tightly regulated. In conjunction, the release of platelet-derived phospholipids and microparticles has also earned merit as synergistic regulators of angiogenesis. Consequently, platelets have been functionally implicated in a range of angiogenesis-dependent processes, including physiological roles in wound healing, vascular development and blood/lymphatic vessel separation, whilst facilitating aberrant angiogenesis in a range of diseases including cancer, atherosclerosis and diabetic retinopathy. Whilst the underlying mechanisms are only starting to be elucidated, significant insights have been established, suggesting that platelets represent a promising therapeutic strategy in diseases requiring angiogenic modulation. Moreover, anti-platelet therapies targeting thrombotic complications also exert protective effects in disorders characterized by persistent angiogenesis.     

Membrane proteins on human megakaryocytes and platelets identified by monoclonal antibodies

We describe five monoclonal antibodies that react with four discrete antigens present on human platelets. Antibodies B2.12 and B59.2 precipitate the glycoprotein IIb-IIIa complex from radiolabeled platelet membrane extracts and inhibit platelet aggregation induced by adenosine diphosphate (ADP), collagen, or epinephrine. The antigen recognized by the two antibodies is present on megakaryocytes but either absent entirely or expressed in small amounts on platelets from Glanzmann's thrombasthenic patients. The antigen recognized by antibody B37.3 is absent from thrombasthenic platelets. Antibody B1.12 reacts with an antigen shared by platelets and 20% of peripheral blood lymphocytes and is a potent inducer of platelet aggregation. Antibody B2.10 reacts specifically with platelets and megakaryocytes but does not affect platelet functions. Thus, these reagents are useful tools in diagnostic and functional studies of both normal and abnormal platelets.     

Golgi proteins in circulating human platelets are distributed across non-stacked,scattered structures

Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown.

Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte.

In conclusion, we suggest that these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research.     

alpha-Actinin and membrane glycoprotein IIIa are different proteins in human blood platelets.

It has been suggested that a platelet protein that is very similar to muscle alpha-actinin is identical to the membrane glycoprotein IIIa (GPIIIa) of platelets and is responsible for anchoring actin filaments directly into the plasma membrane of platelets. To determine if alpha-actinin and GPIIIa are related in platelets, we analyzed the purified proteins on 5% sodium dodecyl sulfate/polyacrylamide gels. The two proteins differ in mobility in both the unreduced and reduced states, and they stain differently with silver stain. In addition, alpha-actinin is a prominent component of the detergent-insoluble cytoskeletons of platelets, whereas GPIIIa is absent from these structures. By using monospecific antisera to the individual proteins, it was demonstrated that alpha-actinin and GPIIIa are immunologically distinct. We conclude that alpha-actinin and GPIIIa are different proteins in human blood platelets and that it is unlikely that alpha-actinin is an integral membrane protein.     

Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6-0.9 M NaCl and at 1-1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000-7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.     

Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions

Proteins secreted by activated platelets can adhere to the vessel wall and promote the development of atherosclerosis and thrombosis. Despite this biologic significance, however, the complement of proteins comprising the platelet releasate is largely unknown. Using a proteomics approach, we have identified more than 300 proteins released by human platelets following thrombin activation. Many of the proteins identified were not previously attributed to platelets, including secretogranin III, a potential monocyte chemoattractant precursor; cyclophilin A, a vascular smooth muscle cell growth factor; calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction, as well as proteins of unknown function that map to expressed sequence tags. Secretogranin III, cyclophilin A, and calumenin were confirmed to localize in platelets and to be released upon activation. Furthermore, while absent in normal vasculature, they were identified in human atherosclerotic lesions. Therefore, these and other proteins released from platelets may contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, as soluble extracellular proteins, they may prove suitable as novel therapeutic targets.     

Complement regulatory proteins in normal human esophagus and esophageal squamous cell carcinoma

BACKGROUND: Altered expression of three complement regulatory proteins, decay-accelerating factor (CD55), membrane cofactor protein (CD46) and homologous restriction factor 20 (CD59) has been identified in human gastrointestinal malignancies, but their expression in esophageal cancer has not been described. Therefore the purpose of the present paper was to study the distribution of these proteins in human normal and malignant esophageal mucosa. METHODS AND RESULTS: In the normal esophageal mucosa, CD55 predominantly stained on the cell membrane of squamous epithelium in the superficial and prickle cell layers, whereas CD46 most intensely stained on the cell membrane in the basal and parabasal cell layers. In contrast to this reciprocal expression of CD55 and CD46, CD59 was broadly distributed on the cell membrane in all layers. In the esophageal squamous cell carcinoma, CD55 staining was intense in the stroma but was negligible in the cancer cells. In contrast, CD46 and CD59 stained almost uniformly on the tumor cell membrane. There was a significant difference in the intensity of the staining of CD55 and CD46 among cells in various layers of normal esophageal mucosa and esophageal carcinoma cells, but not in the staining of CD59. Similar expression patterns of the three complement regulatory proteins in carcinoma cells and in normal epithelium in the basal and parabasal cell layers were observed. CONCLUSIONS: These observations on the expression of the three complement regulatory proteins would help understanding of the host immune responses involving the complement system against esophageal squamous cell carcinoma.     

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.