Abstract. Because ADP has been reported to produce a secondary wave of platelet aggregation in diabetic subjects, and since ADP is known to enhance normal platelet biosynthesis of pro‐aggregating thromboxane A2, we tested whether or not the reported increased sensitivity of diabetic platelets to ADP may also result in increased platelet biosynthesis of thromboxane A2. To test this hypothesis, 14C‐arachidonic acid (14C‐AA) was incubated in vitro with washed human platelets' in the presence or absence of ADP. These studies included platelets isolated from thirty normal volunteers, twenty‐six diabetic subjects with and without known vascular complications, eighteen non‐diabetic pregnant females and fourteen pregnancy‐induced diabetic females. Data from these studies demonstrated: (i) a significant increase in the capability of diabetic platelets in response to ADP to biosynthesize thromboxane A2 from arachidonic acid when compared to platelets from normal controls (P < 0.001); (ii) a significant increase in thromboxane A2 biosynthesis by platelets from pregnancy‐induced diabetic subjects over nondiabetic pregnant females (P < 0.001); (iii) a two‐fold increase in thromboxane A2 biosynthesis by platelets from diabetic subjects with vascular complications when compared to those without vascular complications. Although our data also showed approximately a twofold increase in thromboxane A2 biosynthesis by platelets from diabetic subjects with greater than 10 years of the disease when compared to diabetic subjects with less than 10 years, these latter results were, however, not statistically significant. Results from these studies suggest that a relationship may exist between the markedly increased ADP‐induced platelet aggregation in diabetes mellitus and the vascular complications associated with this disease. Whether or not increased capacity of the diabetic platelet to biosynthesize pro‐aggregating thromboxane A2 in response to ADP or other pro‐aggregating agents is per se a triggering factor in occlusive vascular diseases reported in diabetic subjects must await further studies.

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.

Prostaglandin biosynthesis from arachidonic acid by skin microsomal fraction preparation was enhanced by UV-irradiation at wavelengths of 254 and 360 nm. In the presence of 8-methoxy psoralen (8 MOP) and coal tar, prostaglandin biosynthesis was further enhanced approximately 2-fold by UV-irradiation at 254 nm. Stimulation was less by UV-irradiation at 360 nm. 8-MOP enhanced the conversion of PGE2 into PGF2-9-ketoreductase prepared from skin high speed supernatant fractions. UV-irradiation at 254 nm and 360 nm with or without the photosensitizers had no effect on the activity of the PGE2-9-ketoreductase. These data therefore indicate that the action of UV-irradiation, 8-methoxy psoralen and coal tar on the skin may in part be due to their regulation of the biosynthesis and metabolism of prostaglandins in this tissue.

Human skin membranes bind prostaglandin E2 (PGE2) with high affinity (with an apparent dissociation constant, Kd, of 3.14 X 10(-9) M) and specificity. This binding is inhibited by trypsin or heat treatment suggesting that PGE2 receptors have protein components. Exposure of the membranes to ultraviolet irradiation (UVB) resulted in the loss of the membrane binding capacity for PGE2. This UVB-ihibitory effect could be prevented by 5,5'-dithiobis-(2-nitrobenzoic acid), a known protein sulfhydryl-oxidizing agent and alpha-tocopherol, a known lipid anti-oxidant. These results suggest that UVB-irradiation possibly initiate the reduction of critical protein disulfide groups and the peroxication of lipids in the membranes, which are essential for the receptor-PGE2 interaction.

Specific binding of [3H]prostaglandin E2 ([3H]PGE2) and [3H]prostaglandin F2 alpha ([3H]PGF2 alpha) to plasma membrane and smooth endoplasmic reticulum fractions prepared from rat skin was demonstrated by the Millipore filter assay system. Specific binding was greater in the smooth endoplasmic reticulum fraction. Maximum binding was attained in the presence of Ca++ (0.5 x 10(-3) M), pH 7.2, and at a temperature of 37 C. Unlabeled PGE2 at a concentration of approximately 700 x 10(-9) M inhibited binding to the smooth endoplasmic reticulum fraction by approximately 90%. Other unlabeled prostaglandins, PGE1, PGF2 alpha, and a PGE2 analog, 7-0-13-prostynoic acid, at the same concentration inhibited binding by 40%, 25%, and 30%, respectively. A variety of unlabeled fatty acids (palmitic, oleic, linoleic, eicosatrienoic, and eicosatetraenoic acids), at the higher concentration of approximately 1700 x 10(-9) M, inhibited binding less than 30%, suggesting the presence of specific receptors for PGE2 in the smooth endoplasmic reticulum. Similar results were obtained for the competitive binding studies with [3H]PGF2 alpha. Proteolytic digestion of the membrane fraction by trypsin and pronase, or boiling for 15 min caused marked inhibition of binding, suggesting that the receptors for PGE2 and PGF2 alpha have a protein component. The Scatchard plot analysis of the equilibrium-binding data of [3H]PGE2 and [3H]PGF2 alpha to normal smooth endoplasmic reticulum fractions revealed an apparent dissociation constant (Kd) of 1.1 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 1,0 x 10(-9) M with a binding site concentration of 35 x 10(-12) M for [3H]PGF2 alpha. These data indicate a greater concentration of binding sites for PGE2 than PGF2 alpha in the normal skin smooth endoplasmic reticulum. On the other hand, analysis of smooth endoplasmic reticulum from skin of essential fatty acid-deficient rats revealed a Kd of 1.2 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 2.2 x 10(-9) M with a binding site concentration of 175 x 10(-12) M for [3H]PGF2 alpha. The concentration of binding sites for PGF2 alpha in the smooth endoplasmic reticulum of the skin of these rats is increased 5-fold when compared to normal values, whereas that of PGE2 was not altered. These results suggest a possible alteration of PGF2 alpha specific binding to skin endoplasmic reticulum during the pathophysiological abnormalities that accompany essential fatty acid-deficiency syndrome.