Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha- granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta- thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta- thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.
Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane “ghosts” for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII- detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.
Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000- dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.
Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane “ghosts” for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII- detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.
Diadenosine tetraphosphate (AP4A) is an unusual nucleotide found in a variety of cells, including platelets. It has been suggested that platelet AP4A is stored in the dense granules and is metabolically inactive. We have studied the AP4A content of blood platelets in two patients and three cattle with Chediak-Higashi syndrome (CHS), a hereditary platelet defect with dense granule deficiency. Acid-soluble extractions of whole blood and platelets were neutralized. The adenosine triphosphate (ATP) level was measured by luminescence technique. To measure the AP4A content, the neutralized extract was treated with phosphomonoesterase for removal of ATP. The AP4A content was then measured by coupling the phosphodiesterase and luciferase reaction. The AP4A content was 0.43 nmol/mg protein for normal human platelets and 0.004 nmol/mg protein for CHS platelets. The ATP/AP4A ratio was 67 for normal and 3,023 for CHS platelets. The whole blood AP4A was reduced by 89% in CHS patients who had only a slight decrease in ATP level (26% reduction). Similarly, bovine platelets with CHS showed a marked decrease of AP4A content and a moderate reduction of the ATP level. The platelet ATP/AP4A ratio was 351 and 3,133 for normal and CHS cattle, respectively. Results demonstrate a marked reduction of AP4A in CHS platelets and suggest that AP4A may be a useful marker for the measurement of dense granule content in platelets.
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