David C.B. Mills

Stimulation of human platelet adenylate cyclase by prostaglandin D2

Abstract Prostaglandin D 2 (PGD 2 ) stimulates the formation of cyclic AMP in human platelets measured as the incorporation of radio-activity from previously labelled intracellular nucleotides. In this action it is similar to, and more powerful than PGE 1 . Both inhibition of platelet aggregation and stimulation of cyclic AMP accumulation by PGD 2 and by PGE 1 are potentiated by an inhibitor of platelet phosphodiesterase. A number of minor differences in the response of platelets to PGD 2 and PGE 1 suggest the existence of at least two prostaglandin receptors influencing a single adenylate cyclase.

Metabolism and Function of Human Platelets Washed by Albumin Density Gradient Separation

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium‐free Tyrodes solution. The mean recovery of platelets after two separations relative to plateletrich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [14C]5‐hydroxytryptamine (5HT) in response to adenosine diphosphate (ADP) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP‐induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP‐induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [14C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE1 or PGD2. Platelet coagulant activities were not made available and heparin‐neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37deg;C compared with 25deg;C did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.

The role of thrombin in ADP-induced platelet aggregation and release: a critical evaluation.

Summary. The role of thrombin in ADP‐induced aggregation and release in vitro was critically examined. The addition of heparin or hirudin to citrated platelet rich plasma did not prevent aggregation or release. The addition of citrate to heparinized plasma restored secondary aggregation and release. Hirudin did not prevent irreversible aggregation. These results are incompatible with the hypothesis that thrombin is a primary and necessary mediator of platelet aggregation (Ardlie & Han, 1974; Han & Ardlie, 1974a, b, c). This hypothesis is based in part on the assumption that EDTA enhances the elution of clotting factors from platelets; we found no enhanced elution of factors II, V, X, VIII, IX or XI when platelets were washed in EDTA.

Hereditary Giant Platelet Syndrome

Summary The platelets from two related patients with the hereditary giant platelet syndrome were examined. They were larger than normal but otherwise ultra‐structurally normal; they contained increased storage pools of adenine nucleotides and heparin‐neutralizing activity and took up serotonin at an increased rate. They aggregated normally with ADP and collagen but failed to aggregate with bovine factor VIII and Ristocetin. Some change in shape occurred with ADP, and the reduction in adenylate energy change after addition of ADP to platelet‐rich plasma was smaller than normal.

Distribution of phosphatidylethanolamine arachidonic acid in platelet membranes

Arachidonic acid (20 : 4) and other fatty acids and aldehydes in phosphatidylethanolamine (PE) present on the platelet surface was determined. Surface-exposed PE was isolated by using 2,4,6-trinitrobenzenesulfonate, a non-penetrating probe (Schick, P.K., Kurica, K.B. and Chacko, G.K. (1976) J. Clin. Invest. 57, 1221--1226). PE contains 50% total platelet arachidonic acid. Approx. 16% platelet PE is present on the platelet surface. The study showed that the fatty acid and aldehyde composition of PE on the platelet surface is virtually identical to that in PE present inside the platelet. Therefore, 8 nmol arachidonic acid are present in PE in the outer layer of the plasma membrane in 10(9) platelets.