Victor Skrinska

Thromboxane production and platelet aggregation in diabetic subjects with clinical complications

Abstract The susceptibility of diabetic patients to pathophysiological complications and abnormalities in their platelet function prompted this investigation. A group of 180 insulin-dependent subjects were randomly selected: 38.9% with no other clinical complications; 16.7% with retinopathy; 15.0% - neuropathy; 3.9% - nephropathy; 4.4% - peripheral vascular disease; 21.1% - coronary arterial disease. Twenty seven subjects with no diabetic disease were used as controls. Thromboxane B 2 [stable metabolite of thromboxane A 2 (TXA 2 )], generated by exogenous arachidonic acid and quantitated by electron-capture gas chromatography (EC-GC) was significantly higher in platelets of diabetics with clinical complications than in controls. Platelets in platelet rich plasma (PRP) processed from citrated whole blood of diabetic subjects were more sensitive to ADP- or collagen-induced aggregation than those of non-diabetics. Platelets similarly obtained from diabetics, but separated on agarose gel column rather than obtained in PRP, aggregated with greater sensitivity to arachidonic acid than those of controls. Plasma total cholesterol and triglyceride concentrations were similar in diabetic and non-diabetic subjects. Platelet total phospholipid concentration, percent composition of molecular species and fatty acid distribution were similar in both groups of subjects indicating a similar substrate concentration for prostaglandin biosynthesis. It is postulated that in the platelets of diabetic subjects: a) the observed increase in TXA 2 synthesis is due to the increased activity of the thromboxane synthetase system at one or more sites; b) this increased enzyme activity is the mechanism of greater platelet sensitivity to aggregation.

Platelet Glutathione and Thromboxane Synthesis in Diabetes

The relationship of the reduced glutathione (GSH) content in unstimulated platelets and their capacity to synthesize thromboxane A2 (TXA2), measured by radioimmunoassay of TXB2, was investigated in diabetic and matched control subjects. The GSH content in platelets from diabetic subjects (6.52 ± 0.73 μg/109 platelets, mean ± SD) was significantly (P < 0.001) lower than in platelets from control subjects (10.10 ± 1.58 μg/109 platelets). When platelet-rich plasma (PRP) was stimulated with 1.65 mM arachidonic acid, significantly (P < 0.001) more TXB2 was formed in PRP from diabetic subjects (344 ± 87 ng/2.5 × 108 platelets) than in PRP from control subjects (132 ± 35 ng/2.5 × 108 platelets). Furthermore, the plasma level of TXB2 was increased in diabetic subjects (522 ±117 pg/ml) in comparison with control subjects (187 ± 63 pg/ml). An inverse correlation (r = 0.98) was observed between the GSH content in unstimulated platelets and their capacity to synthesize TXA2 when stimulated with 1.65 mM arachidonic acid. These data suggest that platelet GSH may have an important regulatory effect on platelet TXA2 synthesis and that increased TXA2 synthesis by platelets from diabetic subjects may be the result of low intracellular GSH levels.

Treatment of acute focal cerebral ischemia with prostacyclin.

The object of this investigation was to study the effects of prostacyclin (PGI2) upon the evolution of acute focal cerebral ischemia in the cat. Twenty-five fasted adult cats, lightly anesthetized with nitrous oxide, underwent right middle cerebral artery (MCA) occlusion. Eleven cats received an intracarotid infusion of PGI2 in buffered saline pH 10.5 (100 ng/kg/min at 0.01 ml/kg/min), and 11 cats received intracarotid buffered saline pH 10.5 (0.01 ml/kg/min) without therapeutic agents. Treatment with PGI2 was started upon MCA occlusion and continued for 6 hours. Thirty minutes prior to perfusion, the animals were given fluorescein and Evans blue by intravenous injection. The cats were perfused-fixed in vivo with carbon and buffered formalin 6 hours after MCA occlusion. Another 3 cats received tritium labeled intracarotid PGI2, and peripheral venous samples were collected and assayed for PGI2 plasma levels. Mean arterial pressure was stable in PGI2 treated animals during 6 hours of MCA occlusion, while untreated cats had significant progressive hypertension during that period. The rCBF (measured by the intracarotid 133Xe method) decreased markedly in all animals immediately upon MCA occlusion. However, untreated animals had a significant progressive improvement in rCBF during the occlusion period, while PGI2 treated animals had no such improvement. Quantitative EEG changes, gross edema, areas of fluorescein extravasation, patterns of carbon perfusion, and infarct size were not significantly different in the two groups. While most untreated animals had marked Evans blue extravasation after 6 hours of MCA occlusion, most PGI2 treated animals had no such extravasation, indicating some protection of the blood-brain barrier in these animals.

Isolation of prostacyclin from whole blood

A method was development for the isolation of prostacyclin (PGI2) from whole blood in a fraction suitable for high pressure liquid chromatography (HPLC) separation of PGI2 and 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha). Prostacyclin was stabilized in whole blood by rapidly raising the pH to 10 with Na2CO3 and cooling the samples to 0 degree C. Under these conditions, 2.9% hydrolysis was observed after 20 min. Reverse phase extraction columns were used to directly extract both PGI2 and 6-K-PGF1 alpha from the alkaline plasma with recoveries of greater than 95% using an acetonitrile/2mM Na2B4O7, pH 10, 40/60 elution solvent mixture. An additional 1.7% hydrolysis was found during the column extraction procedure. Final separation of PGI2 and 6-K-PGF1 alpha was performed with HPLC using an alkaline solvent system. This method is capable of rapidly and efficiently extracting and separating PGI2 and 6-K-PGF1 alpha from whole blood or plasma. It introduces less than 5% hydrolysis of PGI2, thus providing a means of applying highly sensitive 6-K-PGF1 alpha assays to the determination of PGI2 levels in physiological samples.

Stability of prostacyclin in human and rabbit whole blood and plasma

The stability of prostacyclin (PGI2) in whole blood and plasma was studied in vitro by measuring the disappearance rate of labeled prostacyclin during a 37 degrees C incubation. Prostacyclin was assayed using a quantitative chromatographic method. The half-life of PGI2 was 6.3 +/- 0.8 minutes (mean +/- s.d., n = 6) in citrated human whole blood, significantly shorter (p less than 0.001) than the 10.7 +/- 2.3 minute half-life in citrated human plasma (n = 7). Prior freezing and thawing of plasma did not affect the rate of PGI2 hydrolysis. These values, including the prolonged half-life in plasma, were similar in the blood (5.4 +/- 1.8 min, n = 7) and plasma (9.0 +/- 1.9 min, n = 14) of diabetic patients. In plasma samples from patients with thrombotic thrombocytopenic purpura, the half-life of prostacyclin (4.9 +/- 1.0 min, n = 4) was significantly shortened (p less than 0.001) compared to that in plasma from normal volunteers. The stability of prostacyclin in rabbit blood and plasma was also quantified. The PGI2 half-life in citrated rabbit plasma (10.8 +/- 1.1 min, n = 3) was similar to that in citrated human plasma from control subjects. In contrast to the findings in human blood, the half-life of PGI2 in citrated rabbit whole blood (11.7 +/- 3.3 min, n = 4) was not different from the rabbit plasma value. Substitution of EDTA for citrate did not affect the half-life in rabbit blood or plasma.

The influence of glutathione and other thiols on human platelet aggregation

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC50 = 0.61 +/- 0.05 mM) was greater than that by cysteine (IC50 = 13 +/- 1 mM) or 6-mercaptopurine (IC50 = 5.4 +/- 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.

Analysis of A, B, E, and F prostaglandins as pentafluorobenzyl esters by electron-capture gas-liquid chromatography

A new and sensitive method is described for the simultaneous analysis of a mixture containing PGE1, PGE2, PGF1alpha, and PGF2alpha by electron-capture gas-liquid chromatography. During derivatization of the mixture, PGE1 and PGE2 were converted to PGB1 and PGB2, respectively, yielding a mixture of PGB1, PGB2, PGF1alpha, and PGF2alpha trimethylsilyl ether pentafluorobenzyl esters. Gas chromatographic resolution of all four derivatives is sufficient for quantitation of each prostaglandin. The A prostaglandins were analyzed by similar conversion to the respective B prostaglandin derivatives. Minimum detection limits for the B and F prostaglandin derivatives were 10 pg and 1 pg, respectively. Samples of rabbit kidney medulla were incubated and analyzed for A, B, E, and F prostaglandins. The results indicate that the method is capable of high recovery and reproducibility.

Behavior of intracellular glutathione during platelet thromboxane synthesis in diabetes

The time-dependent relationship between the levels of the reduced form of glutathione (GSH) and thromboxane A2 (TXA2) synthesis, as measured by the accumulation of TXB2, in platelets from human diabetic and control subjects was investigated during aggregation. In platelets from control subjects, the GSH level decreased to 21% of the initial level within 30 sec in response to arachidonic acid (1.65 mM) and rapidly recovered to 91% by 1 min. In platelets from diabetic subjects, the GSH level decreased to 3% of the initial level within 30 sec and recovered to only 41% by 1 min. During collagen (20 ug/ml) aggregation, platelets from control subjects had a 15 sec lag phase which was followed by a decrease in the GSH level to 21% of the initial level within 1 min and a recovery to 74% by 2 min. Platelets from diabetic subjects in response to collagen showed no lag phase and decreased to 10% of the initial level within 1 min which was followed by a recovery to 34% by 2 min. In all aggregations, the initial GSH level was significantly (p less than .001) lower in platelets from diabetic subjects and remained significantly (p less than .01) lower than GSH in platelets from control subjects throughout the aggregation. The amount of TXB2 formed by platelets from control subjects reached a maximum in response to arachidonic acid and collagen by 1 min and 2 min, respectively, whereas, the TXB2 continued to increase up to 4 min when platelets from diabetic subjects were aggregated. These data indicate that TXA2 synthesis occurs during the decrease in GSH and ceases when the GSH level recovers. The continued synthesis of TXA2 by platelets from diabetic subjects coincides with the gradual recovery of the GSH level.

Treatment of Focal Cerebral Ischemia with Prostacyclin and Indomethacin

An effective medical regimen for the treatment of focal cerebral ischemia has yet to be defined. Therapy ideally should be directed towards improving tissue perfusion, reducing edema, and increasing the tolerance of cerebral tissue, particularly neurons, to the effects of ischemia1. It is unlikely that a single agent will be found to accomplish all these goals. Recent investigations have suggested that progressive microcirculatory impairment5,17,21 and intravascular coagulopathy12 occur in the ischemic brain. Other studies have described increased resistance of pial collateral arteries following experimental middle cerebral artery (MCA) occlusion23. Although it is not known to what extent this microcirculatory impairment contributes to the progression of ischemic changes and neurological damage, it is felt that maintenance of the cerebral microcirculation may enhance the delivery of oxygen and substrate, and potentially allow delivery of other therapeutic agents to the core area of ischemia.