D J Hanahan

Acetylglyceryl ether phosphorylcholine (AGEPC; platelet-activating factor)-induced stimulation of rabbit platelets: correlation between phosphatidic acid level, 45Ca2+ uptake, and (3H)serotonin secretion

When 32Pi-labeled rabbit platelets were incubated with 5 X 10(-10) M 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), either in the presence or absence (0.1 mM EGTA) of added Ca2+, there was a three- to five-fold increase in the [32P]phosphatidic acid (PA) pool within 15 to 20 s. This event was followed by a gradual decrease in the [32P]PA level to near basal level in 5 min. AGEPC effected this change in [32P]PA in a characteristic dose- and time-dependent manner. Polar head group analogs of AGEPC, such as AGEDME and AGEMME, also effected an increase in PA labeling at levels comparable to those previously reported for their activity toward rabbit platelets [K. Satouchi, R. N. Pinckard, L. M., McManus, and D. J. Hanahan (1981) J. Biol. Chem. 256, 4425-4432]. Other analogs, i.e., lysoGEPC and the enantiomer, sn-1-AGEPC, which are inactive toward rabbit platelets, also showed no effect on the level of [32P]PA. The finding that the PA level in rabbit platelets could be manipulated by the addition of AGEPC, without any added Ca2+, provided an excellent model system for establishing a correlation between the uptake of Ca2+, serotonin release, and PA level. Thus, PA must be regarded as a sensitive indicator of a reaction mechanism important to the platelet response to AGEPC, and could be the focal point in promoting calcium uptake during the stimulation process.

The isolation and characterization of a third or neutral phospholipase A2 in the venom of Agkistrodonhalys blomhoffii: An improved fractionation procedure for all three enzymes

The isolation of a new, third phospholipase A2 from Agkistrodon halys blomhoffi is described. On the basis of a pI value of 6.9, it is termed a neutral phospholipase A2. It was characterized as to its amino acid content, activity towards phosphatidylcholine, heat stability, and hemolytic behavior on human erythrocytes. A comparison of these characteristics with those of the acidic and basic phospholipases A2 established the uniqueness of the neutral enzyme. Two particularly important observations were concerned with the complete stability of the three phospholipases on heating at 100 degrees C at pH 6.0 in the presence of 10 mM Ca2+, but variable stability in the absence of Ca2+, and the significant lack of hemolytic activity by the acidic (pI 4.9) phospholipase A2 as compared to the neutral (pI 6.9) and basic (pI 8.7) enzyme which produced extensive hemolysis of human erythrocytes. Other facets of the characteristics of these phospholipases are discussed.

Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin

Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta-aminoethyl ether) N,N-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation.

AGEPC: a potent calcium-dependent chemical mediator in the liver.

AGEPC (i.e. 1-0-alkyl-2-acetyl- sn -glycero-3-phosphocholine) or platelet activating factor is a unique phosphoglyceride first described as a fluid phase mediator of platelet aggregation during immunoglobulin E-induced anaphylaxis in the rabbit (1,2). AGEPC is produced in response to various stimuli in a variety of cells including neutrophils, basophils, monocytes and mast cells (3-7), and is among the most potent mediators formed and released by biological tissues. AGEPC induces aggregation and degranulation of platelets and neutrophils at subnanomolar concentrations (8,9) and is thought to act by interaction with specific receptors (10–13). In platelets, AGEPC-induced aggregation is associated with the turnover of inositol phospholipids, production of phosphatidic acid and protein phosphorylation (12–15). Additional studies have indicated that AGEPC possesses diverse biological actions including contraction of smooth muscles (16–18), negative inotropic cardiac effects (19,20), vasoconstriction (21,22), and exocrine gland stimulation (23). We have recently found that AGEPC possesses a powerful regulatory effect on hepatic metabolism which is the subject of this report.

Dual Effect of Propranolol on the Human Platelet Activation by Thrombin: Potentiation of Free Intracellular Ca2+ Mobilization and Inhibition of Phospholipase D Activity

Propranolol has been employed for a long time in clinical practice as non selective β-adrenoreceptor antagonist. Only the S(−) optical isomer has β-blocking activity, whereas R(+) form is totally ineffective. Early clinical observations (1978–1979) showed that treatment with propranolol improved blood platelet function in patients with cardiovascular diseases. In vitro studies indicated that this compound inhibited human PRP aggregation following exposure to ADP, collagen, epinephrine and Ca2+ ionophore A23187, and diminished aggregation of washed human platelets induced by a low concentration (0.025 U/ml) of thrombin (1). Both S(−) and R (+) isomers as well as (±) racemic form were active. Further studies evaluated the inhibitory effect of propranolol on phosphatidic acid (PA) phosphohydrolase, which converts PA to diacylglycerol in various type of cells (2). It was found that inhibition of PA phosphohydrolase by propranolol led to potentiation of 02 production in neutrophils stimulated with putative receptor agonist fMLP, suggesting a role for PA as a second messenger and for PA phosphohydrolase as a key regulatory enzyme in the interaction between PLC and PLD in the signal transduction (3,4). Sozzani et al., showed recently (5), that both S(−) and R(+) form inhibited protein kinase C (PKC) from human neutrophils and reduced [3H]PDBu binding by neutrophils in the concentration range of 100–200 μM.