Detection in human blood platelets of sialyl Lewis X gangliosides, potential ligands for CD62 and other selectins

Abstract

Activated platelets are known to express P-selectin, a lectin-like adhesion receptor (CD62), through which they bind to sialyl Lewis X (sLex) ligands displayed on the membranes of leukocytes. To determine whether direct platelet-platelet interactions via P-selectin/sLex interactions are also possible, we have examined the ganglioside extract of human blood platelets for the presence of sLex ligands. Using the sensitive method of high-performance thin-layer chromatography (HPTLC)-immunostaining with the monoclonal antibody (mAb) CSLEX or with sialidase followed by mAbs MC480 or PM81, eight sLex bands were demonstrated at Rf 0.01, 0.03, 0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10 chloroform-methanol-aqueous 0.02% CaCl2. The sensitivity of all eight bands to sialidase or endoglycoceramidase confirmed that they were gangliosides. Comparison of the HPTLC mobilities and densities of platelet bands with those from five other human tissues (granulocytes, monoblasts, kidney, aortic endothelium and erythrocytes) in three different solvents revealed three major bands associated with platelets: 3 (Rf0.03), 6 (0.08) and 14 (0.21). Platelet bands were demonstrated not to have resulted from granulocyte contamination. Partial purification of platelet sLex gangliosides by high-performance liquid chromatography and their reaction with 14 oligosaccharide-specific mAbs (FH4, FH5, LM112-161, LM119-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04, SH34, P001 and MC813-70) revealed that band 6 is a multifucosylated neolacto ganglioside and band 14 is a branched, disialo neolacto fucoganglioside. Platelet band 3 combined the features of both bands 6 and 14, and reacted differently than granulocyte band 3. These partial structures resemble gangliosides associated with adhesion in other cell systems. It is concluded that platelets express tissue-specific sLex gangliosides (sLex ligands). Thus, it is possible that platelet-platelet binding may be mediated at least partially through P-selectin/sLex interactions, especially after platelet activation.

The Proteoglycan Lectin Domain Binds Sulfated Cell Surface Glycolipids and Promotes Cell Adhesion

“…On the other hand, selectins and PLDs bind differently to gangliosides, another class of acidic glycolipids containing sialic acids. P-and L-selectins have been shown to bind not only sulfated glycolipids but also gangliosides (37,38), whereas we showed that brevican PLD does not bind gangliosides. This suggests that the mere presence of negative charges is not sufficient for the binding of brevican PLD to sulfated glycolipids.…”

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

“…The sLeX antigen has been detected on the surface of multiple cell types, including granulocytes (particularly neutrophils), monocytes (28,29), platelets (30), natural killer cells (31), activated lymphocytes (32), and helper memory T cells (33), where it may be present on glycoproteins or glycolipids. The sLeX antigen has a critical role in the inflammation process (34).…”

Glycosphingolipid expression in acute nonlymphocytic leukemia: common expression of shiga toxin and parvovirus B19 receptors on early myeloblasts

“…All tissue procurement was carried out with the supervision and approval of the institutional human investigation committee. 49,50 The total GSL fraction of each tissue was isolated using the procedure of Ledeen and Yu with modification. [49][50][51] Briefly, lyophilized cell powders were extracted with chloroform-methanol (C/M 1:1 [vol/vol]; 100 mL/g dry weight) to obtain a total lipid extract.…”

“…49,50 The total GSL fraction of each tissue was isolated using the procedure of Ledeen and Yu with modification. [49][50][51] Briefly, lyophilized cell powders were extracted with chloroform-methanol (C/M 1:1 [vol/vol]; 100 mL/g dry weight) to obtain a total lipid extract. Neutral lipids were isolated by anion exchange chromatography (A-25 DEAE-Sephadex; Sigma, St Louis, MO), saponified with 0.3 N methanolic NaOH to remove phospholipids, followed by dialysis against distilled water.…”

“…49,61 The sample (30.0 mg) was applied to a preparative silica gel column ( [63][64][65] For COSY experiments, 256 to 400 time proportional phase increments (TPPIs) were collected with 32 transients per t 1 , a pulse delay of 4 seconds, and a sweep width of 6000 Hz over 4000 data points in t 2 . For TOCSY and NOESY experiments, 400 TPPIs were collected with 48 transients per t 1 , a pulse delay of 4 seconds, and a mixing time of 100 milliseconds and 500 milliseconds, respectively.…”