H. James Day

Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma

Abstract A method for ATP-ADP determination in plasma by ethanol extraction and firefly luminescence has been modified for platelets. EDTA had to be included in the ethanol for proper inactivation of platelet adenylate kinase. Presence of EDTA inhibited conversion of ADP to ATP (prior to luminescence measurement), presumably through a drop in pH caused by the interaction between H 2 EDTA 2− and the Mg 2+ of the pyruvate-kinase system employed. Introduction of Tris maleate buffer gave 100% ADP-ATP conversion. Optimal amounts of platelet ATP (99%) and ADP (80–90%) were solubilized by 50% EDTA ethanol; less EDTA ethanol failed to inactivate ATP-ADP degrading platelet enzymes whereas increasing EDTA ethanol concentrations gave decreasing solubilization of the nucleotides. ATP and ADP could be extracted from the EDTA ethanol-insoluble material by 0.6 M HClO 4 . ADP and ATP added to such EDTA-ethanol and HClO 4 extracts of both platelet-rich plasma and suspensions of washed platelets gave luminescence responses per mole nucleotide equaling that of ATP-ADP standards. Optimal conditions for nucleotide determination are described as well as the beneficial use of the DuPont luminescence biometer.

Adenine nucleotide metabolism of blood platelets VI. Subcellular localization of nucleotide pools with different functions in the platelet release reaction

Abstract 1. 1. Homogenates of washed human platelets have been fractionated on sucrose gradients, and the amounts of ATP, ADP and serotonin in the fractions have been determined. 2. 2. The bulk (90–95%) of nucleotides was recovered in the soluble fraction, whereas the majority of particle-bound nucleotides was present in a high sucrose density fraction with a ATP ADP ratio of 0.6-1.1. Particle-bound serotonin also had its highest concentration in these particles with an ATP serotonin ratio of 5–7. Incubation of platelet-rich plasma with radioactive adenosine, adenine or orthophosphate gave labeled ATP and ADP in the soluble fraction, membranes and mitochondria, whereas the nucleotides in the high sucrose density particles appeared nonradioactive. 3. 3. When labeled platelets were exposed to collagen, nonradioactive ATP and ADP were released in an ATP ADP ratio equal to that of the granule-bound nucleotides. The amounts of ATP and ADP lost from the cells suggested direct release. During release highly radioactive ATP disappeared and could be recovered intracellularly as IMP. 4. 4. Thrombin released maximally 60% of platelet ATP+ADP in an ATP ADP ratio of 0.78. About 20% ATP disappeared without being released. 5. 5. The results can be explained by assuming 3 pools of ATP+ADP in platelets: One pool (60% of total ATP+ADP, ATP ADP = 0.6−1.1 ) is stored in special granules, does not participate in metabolism and is directly extruded during the release reaction. A second pool consists presumably of ATP only (10% of total ATP+ADP), and is consumed intracellularly during release and converted to IMP. A third pool (30% of total ATP+ADP) present in cytoplasm, mitochondria and membranes is unchanged during release and is in equilibrium with the second pool.

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.

Adenine nucleotide metabolism of blood platelets V. Subcellular localization of some related enzymes

Abstract 1. 1. Subcellular localization of adenosine kinase, adenosine deaminase, adenine phosphoribosyltransferase, hexokinase, pyruvate kinase and adenylate kinase in human platelets has been studied with sucrose gradient centrifugation of platelet homogenates and has been compared with the localization of lactate dehydrogenase, cytochrome c oxidase and 4 acid hydrolytic enzymes. 2. 2. Adenosine kinase was probably associated with both the membrane and combined mitochondria-α-granule fractions, whereas adenosine deaminase, adenine phosphoribosyl transferase and pyruvate kinase were almost completely solubilized during homogenization. 3. 3. More than half the platelet hexokinase activity was bound to particles and revealed a mixed membraneous-mitochondrial distribution pattern. Two thirds of the platelet adenylate kinase were associated with particles from the membrane, combined mitochondria-α-granule and dense α-granule fractions. Its distribution pattern did not resemble any of those for other enzymes.

Introduction to the Brook Lodge Workshop on Animal and Human Models in Thrombosis Research

Remarkable progress has been made in the field of thrombosis research in the past decade. The platelet has been shown to have an important part not only in hemostasis but also in the pathogenesis of thromboembolic disorders. Much has been learned about platelet-vessel wall interactions and such factors as lipids and prostaglandins in promoting hemostasis and possibly thrombosis. That more should be learned about the initial reactions — those involving platelets and the vessel wall — in the genesis of a thrombus seems particularly important because their regulation can offer new opportunities to prevent thromboembolic disease.