Platelet glycohydrolase activities: Characterization and release

Abstract

Granules containing acid hydrolases have been detected in human platelets but have not been thoroughly characterized. We have studied the activity and characteristics of glycohydrolases present in normal human platelets, evaluated their release upon stimulation with thrombin, and assessed the contribution of platelet - released lysosomal contents to the glycohydrolase activity present in normal serum. Platelets contained a remarkable glycohydrolase activity with a prevalence of beta - N-acetylhexosaminidase. All glycohydrolases were released to some extent upon stimulation with thrombin and contributed to the glycohydrolase activity found in human serum. alpha-Mannosidase and alpha-galactosidase were partially inactivated after release by a mechanism as yet undefined. In addition, thrombin stimulation affects the intraplatelet isoenzyme pattern of beta-N-acetylhexosaminidase by producing the appearance of a new form.

Defective plateletβ-N-acetyl hexosaminidase content and release in chronic myeloproliferative disorders

“…One of the most consistent defects so far reported is the reduction in the content and release of and granules, in part due to an in vivo leakage (platelet 'exhaustion') [15][16][17][18]. Human platelets contain and release in vivo upon activation, besides -and -granules, also -granules [26], which store lysosomal enzymes, particularly acid glycohydrolases, the most abundant of which is Hex [25]. No studies have previously examined whether the platelet Hex content and in vivo release is altered in MPD patients.…”

“…DEAE-cellulose chromatography was performed as previously described [25,32] using a 1-ml column equilibrated with 10 mM sodium phosphate buffer, pH 6.0. Enzymatic activity retained by the column was eluted in a linear gradient of NaCl that reached a concentration of 0.5 M in 20 ml of buffer, then washed with 5 ml of 1 M NaCl.…”

“…Hex activity was measured as previously described [25] using 3 mM 4-methylumbelliferyl--N-acetylglucosaminide (4-MUGlcNac) in 0.1 M citric acid/0.2 M disodium phosphate buffer, pH 4.5. Protein content was determined by the Bradford method [31] using crystalline bovine gamma globulin as standard.…”

“…Lysosomes, and in particular their most abundant enzyme -N-acetylhexosaminidase (Hex, EC 3.2.1.5.2), can be released by platelets in vitro, upon stimulation with strong agonists such as thrombin [24,25], and we have previously shown that platelets release their lysosomal content in vivo too, in healthy humans, during activation [26]. No studies, however, have analysed the content, release and isoenzymatic pattern of platelet Hex in vivo in patients with myeloproliferative disorders.…”

Assay of Serum/Plasma β-N-Acetylhexosaminidase Isoenzymes by Heat Inactivation Using a Continuous Spectrophotometric Method Adapted to a Centrifugal Analyzer

“…Likewise, these authors find significantly higher ß-7V-acetylhexosaminidase activity in serum than in plasma, and suggest that the release of enzyme from the platelets could occur during coagulation. Recently, Emiliani et al (33) have demonstrated that stimulation with thrombin provokes a release by platelets of ß-Af-acetylhexosaminidase and other glycohydrolases.…”