Platelets, human radiolabeling

Fig. 8.8. Human platelets after radiolabeling, examined by scanning electron microscopy. No sign of morphological alteration is visible. Perfusion fixation, critical point drying, x8000...

Fig. 8.9. Human platelets after the radiolabeling procedure, examined by transmission electron microscopy. Almost no signs of platelet activation, manifested as pseudopodia formation. Perfusion fixation, critical point drying, x 40 500...

Wagner X, Granegger S, Dembinska-Kiec A, Sinzinger H (1998) Nitric oxide (NO) for radiolabelling of human platelets. In Sinzinger H, Thakur ML (eds) Nuclear medicine research. Facultas, Vienna, p 58... 

Static Adsorption of Plasma Proteins on Glass. Initial studies of the interaction of proteins with artificial surfaces concerned the highly simplified situation of static adsorption on glass from solutions of purified radiolabeled human plasma proteins. Albumin was chosen as a major plasma protein known for its non thrombogenic properties (5>6). Fibrinogen and fibronectin, on the contrary, are major proteins of plasma which enhance platelet and cellular adhesion (4.5.7.23-25). 

In showing that the release of arachidonic acid was selective. Bills el al7 had studied the incorporation of several radiolabelled fatty acids into the complex lipids of human platelets and the subsequent fate of the radiolabel after stimulation of the platelets with thrombin. They found a dramatically greater loss from phosphatidylcholine of radioactive arachidonic acid compared with the other radiolabelled fatty acids. By contrast, Mahadevappa and Holub observed decreases in all the molecular species of phosphatidylcholine after thrombin treatment of platelets prelabelled with radioactive glycerol. They concluded that arachidonate is not selectively released from this phospholipid because the results obtained with radioactive fatty acids were due to unique patterns of incorporation, while those obtained with radioactive glycerol represented the endogenous phospholipid pool. 

The other sources of the arachidonic acid released by platelets in response to thrombin are phosphatidylcholine and phosphatidylethanolamine (Table 1.1). However, each of these phospholipids exists as three species in platelets, namely as the diacyl-, 1-0-alkyl-, or 1-0-alk-l enyl- forms (Table 1.2). Table 1.2 also shows that l-O-alkyl-2-acyl-PC and l-O-alk-l enyl-2-acyl-PE are enriched in arachidonic acid relative to the other classes of PC or PE, giving rise to the idea that they could serve as important sources of arachidonic acid in stimulated platelets. Purdon and Smith " prelabelled platelets with pH]-arachidonic acid and followed the changes in the different species of PC and PE following thrombin stimulation. It was found that while there was a decrease in radiolabel of both 1,2-diacyl-PC and 1,2-diacyl-PE at all times studied, there was no decrease in the other species of PC or PE, and, indeed, radiolabel in l-O-alkyl-2-acyl-PC and l-0-alk-l -enyl-2-acyl-PE increased at later time points (3-5 min) after thrombin. The thrombin-induced incorporation of arachidonic acid in plasmalogen PE was observed previously by others. Purdon and Smith " concluded that, upon stimulation of human platelets, arachidonic acid is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by cyclo-oxygenase and lipoxygenase, while certain other pools of phosphatidylcholine and phosphatidylethanolamine act to collect arachidonic acid. 

Purdon, A.D., Patelunas, D. and Smith, J.B. (1987). Resolution of radiolabelled molecular species of phospholipid in the human platelets. Lipids 22, 116-120... 

There is little information on prostaglandin formation by human cardiac tissue, most of which, based on measurements of thromboxane B2, prostaglandin E2 and 6-keto-prostaglandin in aortic and coronary sinus blood, is likely to be highly artifactual. In a recent experiment, Nowak and coworkers infused radiolabelled arachidonic acid into the coronary arteries of male volunteers 3 days following a single dose of aspirin, 600 mg, by which time cyclo-oxygenase activity in endothelial cells, but not in platelets, would... 

The half-life of thromboxane A2 in platelet-poor plasma was determined as described (9). Briefly, radiolabelled thromboxane A2 was generated from human platelets as enzymatic source and immediately dived in the studied plasma. Then, aliquots of plasma were decanted in 80 volumes of methanol to obtain mono-o-methyl thromboxane B2 from thromboxane A2 (10). Derivatized thromboxane B2 was radiochemically assayed (8) and a semi-logarithmic regression leads to half-life determination. 

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