Platelet Activation-dependent Granule-external Membrane Research Articles

Platelet Activation Induced by Combined Effects of Anticardiolipin and Lupus Anticoagulant IgG Antibodies in Patients with Systemic Lupus Erythematosus

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p<0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+ x LA+ plasma only, but was significantly augmented by aCL+ x LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+ x LA-, aCL- x LA+ and aCL- x LA- plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+ x LA+ plasma (aCL+ x LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+ x LA--IgG and aCL- x LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.

Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes.

The isolation and structural characterization of fucosylated neolacto-series gangliosides with linear poly-N-acetyllactosaminyl chains from normal human granulocytes is described. Gangliosides were purified by consecutive use of anion exchange HPLC on Fractogel TMAE-650(S), adsorption and reversed phase HPLC on Nucleosil 50-7 and Nucleosil 7C18 columns, respectively. TLC immunostaining with carbohydrate specific monoclonal antibodies, fast atom bombardment-mass spectrometry (FAB-MS) of the permethylated derivatives and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates were used for structure elucidations. One ganglioside was identified as sialyl Lewis(x) antigen with nLcOse6Cer core, Neu5-Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc NAc beta 1-3 Gal beta 1-4Glc beta 1-1Cer. Furthermore, monosialylated ceramide deca-, undeca-, dodeca- and tridecasaccharides with three (nLcOse8Cer) and four (nLcOse10Cer) linear lactosaminyl repeats were identified, carring one to three fucoses. The ceramide portions were found to contain C18 sphingosine and predominantly C16:0 fatty acids. All monosialogangliosides were homogenous concerning their terminal alpha 2-3 Neu5Ac-sialylation, but different in their fucosylation status. Beside VI3Neu5Ac, V3Fuc-nLcOse6Cer, in two of the fucosylated polylactosaminyl ganglioside fractions the sialyl Lewis(x) epitope was found, whereas five species expressed the terminal VIM-2 motif. The role of protein linked sialy Lewis(x) epitope of human granulocytes as a ligand for endothelial leukocyte adhesion molecule-1 (ELAM-1; E-selectin) and platelet activation-dependent granule external membrane protein (PADGEM; P-selectin) is well documented. However, the involvement of endothelial cells E-and/or P-selectin mediated cell-cell adhesion via lipid bound sialyl Lewis(x) and/or VIM-2 epitopes on human granulocytes has to be proved in further investigations.

Plasma selectin levels in patients with Graves' disease.

Adhesion molecules relate to cell invasion of autoimmune thyroid disease. We studied plasma soluble P-Selectin (platelet activation-dependent granule-external membrane protein), E-Selectin (endothelial leukocyte adhesion molecule) and L-Selectin (leukocyte endothelial cell adhesion molecule-1) levels in patients with Graves' disease before and during methimazole treatment. Plasma P-, E- and L-Selectin levels in patients with untreated Graves' disease were significantly higher than those in normal subjects. Plasma P-Selectin levels decreased when their thyroid functions were normal for more than 6 months after the start of methimazole treatment. No significant change in plasma E- and L-Selectin levels in patients with Graves' disease was found between hyperthyroid state and euthyroid state after the start of methimazole treatment, but plasma L-Selectin levels in patients with untreated Graves' disease were significantly lower than those in the patients in the first euthyroid state. There was no significant correlation between plasma P-Selectin levels and serum FT4 levels, nor between plasma P-Selectin levels and serum FT3 levels. These results suggested that thyroid hormones might reflect expression of P-, L- and E-Selectin from endothelial cells, or lymphocytes, or platelets in patients with Graves' disease.

Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies

During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the alpha-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15,000 to 20,000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11,000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages.

Inhibition of platelet functions by a monoclonal antibody (LYP20) directed against a granule membrane glycoprotein (GMP-140/PADGEM)

Granule membrane protein (GMP-140), also known as platelet activation- dependent granule-external membrane (PAD-GEM) is an integral membrane glycoprotein that is expressed on the platelet surface following degranulation. GMP-140, also expressed by endothelial cells, is part of a new family of cell adhesion molecules (selectins) related to the endothelial leukocyte adhesion molecule (ELAM-1) and to the lymphocyte homing receptors in humans (Leu-8/TQ1) and in mouse (gp90MEL-14). The role of GMP-140 in platelet functions remains to be elucidated. In this study, a monoclonal antibody, LYP20, was raised against GMP-140. LYP20, directed against a disulphide bridge-dependent epitope, significantly binds to thrombin-stimulated platelets (12,200 +/- 1,184 bound molecules/platelet, kd = 5.0 +/- 0.61 nmol/L) compared with controls (2,400 +/- 266 molecules/platelet, kd = 2.3 +/- 0.54 nmol/L) and inhibits collagen or thrombin-induced aggregation of washed platelets or platelets in platelet-rich plasma. In addition, LYP20 inhibits rosetting of thrombin-activated platelets to U937 cells. These results strongly suggest that GMP-140 plays an important role in platelet aggregation and platelet interaction with other blood cells.

Inhibition of Platelet Functions by a Monoclonal Antibody (LYP20) Directed Against a Granule Membrane Glycoprotein (GMP-140/PADGEM)

AbstractGranule membrane protein (GMP-140), also known as platelet activation-dependent granule-external membrane (PAD-GEM) is an integral membrane glycoprotein that is expressed on the platelet surface following degranulation. GMP-140, also expressed by endothelial cells, is part of a new family of cell adhesion molecules (selectins) related to the endothelial leukocyte adhesion molecule (ELAM-1) and to the lymphocyte homing receptors in humans (Leu-8/TQ1) and in mouse (gp90MEL-14). The role of GMP-140 in platelet functions remains to be elucidated. In this study, a monoclonal antibody, LYP20, was raised against GMP-140. LYP20, directed against a disulphide bridge-dependent epitope, significantly binds to thrombin-stimulated platelets (12,200 ± 1,184 bound molecules/platelet, kd = 5.0 ± 0.61 nmol/L) compared with controls (2,400 ± 266 molecules/platelet, kd = 2.3 ± 0.54 nmol/L) and inhibits collagen or thrombin-induced aggregation of washed platelets or platelets in platelet-rich plasma. In addition, LYP20 inhibits rosetting of thrombin-activated platelets to U937 cells. These results strongly suggest that GMP-140 plays an important role in platelet aggregation and platelet interaction with other blood cells.

PADGEM-dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF III (CD15)

PADGEM (platelet activation-dependent granule-external membrane protein) is a leukocyte receptor of activated platelets that mediates cellular adhesion of platelets to neutrophils and monocytes. To identify the natural ligand on neutrophils and monocytes that interacts with PADGEM, we have evaluated anti-leukocyte antibodies for their ability to block leukocyte-PADGEM binding. Only anti-CD15 antibodies were able to inhibit the binding of neutrophils, monocytes, HL60 cells, and U937 cells to platelets. Anti-CD15 antibodies inhibited the binding of U937 cells to PADGEM-expressing COS cells and to purified PADGEM incorporated into phospholipid vesicles. The CD15 antigen, lacto-N-fucopentaose III (Galβ1→4[Fucα1→3]NAcGlcβ1→3Gal-β1→4Glc), inhibited the interaction of neutrophils or HL60 cells with platelets, whereas lacto-N-fucopentaose I did not; lacto-N-fucopentaose II demonstrated minimal inhibition. Lacto-N-fucopentaose III, and to a lesser extent lacto-N-fucopentaose II, but not lacto-N-fucopentaose I, inhibited the interaction of HL60 cells with COS cells transfected with PADGEM cDNA. CD15, lacto-N-fucopentaose III or Le x, is a component of the PADGEM ligand on neutrophils and monocytes.

PADGEM protein: A receptor that mediates the interaction of activated platelets with neutrophils and monocytes

PADGEM (platelet activation dependent granule-external membrane protein) is an integral membrane protein of the alpha granules of platelets and Weibel-Palade bodies of endothelial cells that is expressed on the plasma membrane upon cell activation and granule secretion. Activated platelets, but not resting platelets, bind to neutrophils, monocytes, HL60 cells, and U937 cells. This interaction is inhibited by anti-PADGEM antibodies, PADGEM, and EDTA; anti-GPIlb-Illa, anti-thrombospondin, anti-GPIV, and thrombospondin produce no effect. Neutrophils and U937 cells, in contrast to Jurkatt cells, contain PADGEM recognition sites, as shown by binding of PADGEM contained in phospholipid vesicles. These results indicate that PADGEM mediates adhesion of activated platelets to monocytes and neutrophils. Therefore, PADGEM shares not only structural but also functional homology with ELAM-1 and MEL-14, members of a new family of vascular cell adhesion molecules.

Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets.

The activated platelet is a potential target for the localization of thrombi in vivo since, after stimulation and secretion of granule contents, activated platelets are concentrated at sites of blood clot formation. In this study, we used antibodies specific for a membrane protein of activated platelets to detect experimental thrombi in an animal model. PADGEM (platelet activation-dependent granule-external membrane protein), a platelet alpha-granule membrane protein, is translocated to the plasma membrane during platelet activation and granule secretion. Since PADGEM is internal in unstimulated platelets, polyclonal anti-PADGEM and monoclonal KC4 antibodies do not bind to circulating resting platelets but do interact with activated platelets. Dacron graft material incubated with radiolabeled KC4 or anti-PADGEM antibodies in the presence of thrombin-activated platelet-rich plasma bound most of the antibody. Imaging experiments with 123I-labeled anti-PADGEM in baboons with an external arterial-venous Dacron shunt revealed rapid uptake in the thrombus induced by the Dacron graft; control experiments with 123I-labeled nonimmune IgG exhibited minimal uptake. Deep venous thrombi, formed by using percutaneous balloon catheters to stop blood flow in the femoral vein of baboons, were visualized with 123I-labeled anti-PADGEM. Thrombi were discernible against blood pool background activity without subtraction techniques within 1 hr. No target enhancement was seen with 123I-labeled nonimmune IgG. 123I-labeled anti-PADGEM cleared the blood pool with an initial half-disappearance time of 6 min and did not interfere with hemostasis. These results indicate that radioimmunoscintigraphy with anti-PADGEM antibodies can visualize thrombi in baboon models and is a promising technique for clinical thrombus detection in humans.

A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein.

We have identified and purified a platelet integral membrane protein (140,000 mol wt), using the KC4 monoclonal antibody specific for activated platelets, that is internal in resting platelets but exposed on activated platelets (Hsu-Lin S.-C., C.L. Berman, B.C. Furie, D. August, and B. Furie, 1984, J. Biol. Chem. 259: 9121-9126.). The expression of the protein on the platelet surface is secretion-dependent. This protein has been named platelet activation-dependent granule-external membrane (PADGEM) protein. PADGEM protein is distinct from the surface glycoproteins of resting platelets, but identical to the S12 antigen, GMP-140. Using immunofluorescent staining, resting platelets failed to stain for PADGEM protein with the KC4 antibody, but after permeabilization showed a punctate staining of the cell interior. Thrombin-stimulated intact platelets stained with a peripheral rim pattern thus demonstrating the translocation of PADGEM protein from an internal location to the cell surface. PADGEM protein expression on the platelet surface at varying thrombin concentrations correlated with alpha granule release, as measured by the secretion of platelet factor 4. Further evidence for an alpha granule localization of PADGEM protein was provided by nitrogen cavitation of resting platelets followed by metrizamide density gradient centrifugation; PADGEM protein codistributed with platelet factor 4. Using immunoelectron microscopy, the protein was localized to the alpha granule in frozen ultrathin sections of resting platelets labeled using rabbit anti-PADGEM protein antibodies, whereas in thrombin-activated platelets, the plasma membrane was labeled. These studies indicate that PADGEM protein is a component of the alpha granule membrane of resting platelets and is incorporated into the plasma membrane upon activation and secretion.