Thrombin-induced Secretion Research Articles

Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid

The lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was biosynthesized, purified and incubated with washed human platelets. It inhibited arachidonic acid, azo-prostaglandin H 2 or U-46619-induced aggregation and secretion in a concentration-dependent fashion (IC 50 = 2–3 μM. Collagen-induced aggregation and secretion were also inhibited (IC 50 = 6 μM. 12-HPETE inhibited malondialdehyde and thromboxane B 2 formation in platelets stimulated with arachidonic acid or thrombin. While thrombin-induced aggregation was unaffected by 50 μM 12-HPETE, thrombin-induced secretion was inhibited. Inhibition of secretion by 12-HPETE was observed in platelets from untreated as well as aspirin-treated donors, indicating that 12-HPETE inhibits secretion independently of its ability to inhibit prostaglandin formation. Aggregation of washed human platelets by arachidonic acid yielded a bell-shaped concentration-response curve. Diminished aggregation at higher concentrations was associated with an increase in the ratio of lipoxygenase products to thromboxane B 2. The data suggest that 12-HPETE formation may regulate platelet aggregation and secretion and that its primary effect, at low concentrations, is inhibition of endoperoxide-induced responses. At higher concentrations 12-HPETE also inhibits arachidonic acid metabolism. Thus, the combined inhibitory effects on endoperoxidase-induced aggregation and thromboxane formation would explain the dimineshed aggregation observed in response to high concentrations of arachidonic acid.

Human Platelet Surface Binding of Endogenous Secreted Factor VIII-von Willebrand Factor and Platelet Factor 4

Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with 125I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which the platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.

Human platelet surface binding of endogenous secreted factor VIII-von Willebrand factor and platelet factor 4

Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with 125I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which the platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.

Inhibition of Thrombin-Induced Secretion from Platelets by Chlortetracycline and Its Analogs

Chlortetracycline (CTC) (1.0 mM) blocks platelet secretion after a few seconds preincubation. The amount needed for inhibition can be reduced relative to time of preincubation. 50 micro M CTC. Two tetracycline analogs, anhydrotetracycline and demeclocycline (DMC), have different solubility properties in nonionic medium, but inhibit secretion at the same concentration, with little effect on the metabolic ATP level. The results suggest that CTC and its analogs do not inhibit platelet function by acting as metabolic inhibitors. While CTC causes increased leakage of cytoplasmic content at the concentrations where secretion is blocked more than 90%, DMC doses not cause leakage even at much higher concentration, so that there seems to be no connection between the induction of leakage (i.e. the membrane-active properties) and the inhibitory effect of the drugs.

Glanzmann's thrombasthenia: Deficient association of actin with the platelet membrane following thrombin-induced secretion

Glanzmann's thrombasthenia: Deficient association of actin with the platelet membrane following thrombin-induced secretion

Effect of three calcium antagonists on platelet secretion and metabolism

Three compounds, 8-( N, N-diethylamino-octyl 3,4,5-trimethoxybenzoate, HCl (TMB-8), 2-propyl-3-dimethylamino-5,6-methylenedioxyindene HCl (2-PIA), and chlortetracycline, were investigated to determine whether their effects on washed human platelets were compatible with a suggested role as calcium antagonists. TMB-8 had little effect on levels of metabolic ATP and IMP, whereas chlortetracycline caused a decrease in metabolic ATP and an increase in IMP; both compounds inhibited thrombin-induced secretion and changed the platelets to spheres. At concentrations up to 0.5 mM, 2-PIA caused a decrease in ATP and an increase in IMP, an induction of secretion, and a centralization of electron-dense material in the platelets, all changes which suggest induction of secretion. The ultrastructural, functional and metabolic effects of TMB-8 and the type of interference by the drug with the effects of thrombin and 2-PIA on the same variables suggest that TMB-8 is mainly a membrane-active drug. Chlortetracycline on the other hand caused changes in platelet metabolism, ultrastructure, and function which, at least partly, indicate an effect on intracellular mechanisms. Both TMB-8 and 2-PIA, and to lesser degree chlortetracycline, caused loss of cytoplasmic nucleotides from the platelets.

Membrane Changes Associated with Platelet Activation

The effect of aggregation and secretion on membrane proteins was studied in washed human platelets. Reversible aggregation without secretion was stimulated by ADP and secretion without aggregation was stimulated by thrombin in the presence of EDTA. No loss of platelet surface glycoproteins occurred during reversible ADP-induced platelet aggregation, as measured by quantitative polyacrylamide gel electrophoresis analysis of platelets that were labeled with (125)I-diazotized diiodosulfanilic acid (DD(125)ISA) before ADP stimulation. Also, no new proteins became exposed on the platelet surface after ADP aggregation, as determined by DD(125)ISA labeling after stimulation. Thrombin-induced platelet secretion also caused no loss of platelet surface glycoproteins. However, after platelet secretion two new proteins were labeled by DD(125)ISA: (a) actin and (b) the 149,000-mol wt glycoprotein (termed GP-G), which is contained in platelet granules and secreted in response to thrombin. The identity of DD(125)ISA-labeled actin was confirmed by four criteria: (a) comigration with actin in three different sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, (b) elution from a particulate fraction in low ionic strength buffer, (c) co-migration with actin in isoelectric focusing, and (d) binding to DNase I. The identity of actin and its appearance on the platelet surface after thrombin-induced secretion was also demonstrated by the greater binding of an anti-actin antibody to thrombin-treated platelets, measured with (125)I-staphylococcal protein A.Therefore, major platelet membrane changes occur after secretion but not after reversible aggregation. The platelet surface changes occurring with secretion may be important in the formation of irreversible platelet aggregates and in the final retraction of the blood clot.

The mechanism of thrombin-induced platelet factor 4 secretion

We have measured thrombin-induced secretion of platelet factor 4 antigen (PF4) and simultaneously followed its intracellular translocation by immunofluorescence. In permeable resting platelets, speckled intracellular immunofluorescent staining for PF4 was observed. Addition of thrombin to washed platelets at 22 degrees C resulted in secretion of PF4 and formation of large (approximately 0.5 micrometer) immunofluorescent masses. These masses moved to the cell periphery during secretion and were virtually absent at the conclusion of secretion. Ultrastructural examination of thrombin-treated platelets revealed vacuoles corresponding in size, shape, and time of occurrence to the large immunofluorescent masses of PF4. These vacuoles contained PF4 by immunoferritin staining of frozen thin sections; they therefore appear to represent the ultrastructural counterpart of the large PF4 masses. When intact cells were stained for PF4 after thrombin addition, only 5.6% of the large masses stained. Thus, during secretion, PF4 antigen is consolidated into large closed pools that appear as vacuoles in the electron microscope.

The Mechanism of Thrombin-Induced Platelet Factor 4 Secretion

We have measured thrombin-induced secretion of platelet factor 4 antigen (PF4) and simultaneously followed its intracellular translocation by immunofluorescence. In permeable resting platelets, speckled intracellular immunofluores-cent staining for PF4 was observed. Addition of thrombin to washed platelets at 22°C resulted in secretion of PF4 and formation of large (~0.5 μm) immunoflourescent masses. These masses moved to the cell periphery during secretion and were virtually absent at the conclusion of secretion. Ultrastructural examination of thrombin-treated platelets revealed vacuoles corresponding in size, shape, and time of occurrence to the large immunofluorescent masses of PF4. These vacuoles contained PF4 by immunoferritin staining of frozen thin sections; they therefore appear to represent the ultrastructural counterpart of the large PF4 masses. When intact cells were stained for PF4 after thrombin addition, only 5.6% of the large masses stained. Thus, during secretion, PF4 antigen is consolidated into large closed pools that appear as vacuoles in the electron microscope.

Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides.

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [(14)C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [(14)C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected (14)C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, beta-N-acetylglucosaminidase, beta-glucuronidase and beta-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)-response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.