Francesca Catella

Platelet Activation in Unstable Coronary Disease

Pathological and clinical studies have suggested that platelets have a role in the pathogenesis of unstable angina and myocardial infarction. However, the relation of platelet activation to episodic ischemia in patients with unstable angina is unknown. We assessed the biosynthesis of thromboxane and prostacyclin as indexes of platelet activation in patients with stable and unstable coronary disease by physicochemical analysis of metabolites in plasma and urine. Prostacyclin biosynthesis was markedly elevated in patients with acute myocardial infarction and correlated with plasma creatine kinase (r = 0.795; P less than 0.001). The largest rise in thromboxane synthesis was observed in patients with unstable angina, in whom 84 percent of the episodes of chest pain were associated with phasic increases in the excretion of thromboxane and prostacyclin metabolites. However, 50 percent of such increases were not associated with chest pain, possibly reflecting silent myocardial ischemia. These data indicate that platelet activation occurs during spontaneous ischemia in patients with unstable angina. The increment in prostacyclin biosynthesis during such episodes may be a compensatory response of vascular endothelium that limits the degree or effects of platelet activation. If so, biochemically selective inhibition of the synthesis or action of thromboxane A2 would be desirable in the treatment of unstable angina. In contrast, thromboxane inhibitors or antagonists would not be expected to be effective in patients with chronic stable angina, in whom there was no increase in the formation of thromboxane A2.

Marked platelet activation in vivo after intravenous streptokinase in patients with acute myocardial infarction.

We assessed thromboxane biosynthesis as an index of platelet activation in 6 patients with acute myocardial infarction receiving intravenous streptokinase. Urinary 2,3-dinor-thromboxane B2 and plasma 11-dehydro-thromboxane B2, major enzymatic metabolites of thromboxane A2, were markedly increased after intravenous streptokinase (11,063 +/- 2758 pg/mg creatinine and 33 +/- 10 pg/ml, respectively) compared with levels in patients not receiving thrombolytic therapy (502 +/- 89 pg/mg creatinine and 3 +/- 0.7 pg/ml). Prostacyclin biosynthesis also increased markedly after streptokinase coincident with the increase in thromboxane A2 formation. Administration of aspirin between the time of onset of coronary thrombosis and reperfusion both in man and in a canine preparation demonstrated that this reflected thromboxane biosynthesis de novo and not metabolism of preformed inactive thromboxane B2 washed out from the coronary circulation. Since the platelet is the major source of thromboxane A2, these findings suggest that there is marked platelet activation after coronary thrombolysis with streptokinase. Studies in vitro demonstrated that streptokinase enhanced platelet activation in a dose-dependent manner, resulting in the secondary release of thromboxane A2. The increase in platelet activation and thromboxane A2 biosynthesis may limit the therapeutic effect of intravenous streptokinase in acute myocardial infarction.

Paired analysis of urinary thromboxane B2 metabolites in humans

11-Dehydro-TxB2 and 2,3-dinor-TxB2 are products of the two major pathways of thromboxane metabolism in man. In this study we compared urinary excretion of 2,3-dinor-TxB2 and 11-dehydro-TxB2 as indices of Tx biosynthesis in vivo. We performed three studies to assess i) the relative abundance of these two metabolites in the urine of healthy subjects, ii) their cellular origin under physiological conditions and iii) their relative formation during platelet activation. In healthy normal volunteers urinary 11-dehydro-TxB2 is more abundant than 2,3-dinor-TxB2 (792 +/- 119 pg/mg creatinine vs 106 +/- 21 pg/mg creatinine). Administration of a dose of aspirin selective for platelet cyclooxygenase (20 mg/day for 10 days) caused substantial and comparable suppression of both 11-dehydro-TxB2 (mean 82 +/- 4.9%) and 2,3-dinor-TxB2 (mean 79 +/- 6.9%). recovery of excretion of both metabolites after a nonselective aspirin regimen (325 mg BID for 3 days) corresponded to platelet life-span. Furthermore, excretion of both metabolites was increased in patients with severe atherosclerosis consistent with the known increase in platelet activation in this setting. Quantitative analysis of both urinary 11-dehydro-TxB2 and 2,3-dinor-TxB2 by GC-MS established that, in contrast to previous assumptions, 11-dehydro-TxB2 is the most abundant urinary metabolite of TxB2. The aspirin study demonstrates that platelets are the major source of both metabolites in urine, consistent with their increased excretion in severe atherosclerosis. Combined analysis of both metabolites will distinguish altered metabolism from increased biosynthesis of thromboxane A2.

Radioimmunoassay of 11-dehydrothromboxane B2 in human plasma and urine

Because of the discrepancy between the capacity of platelets to synthesize thromboxane B2 ex vivo and the actual synthetic rate in vivo, measurement of thromboxane B2 in plasma is highly influenced by sampling-related artifacts. We have developed and validated a radioimmunoassay for a major enzymatic derivative of thromboxane B2 with an extended plasma half-life, i.e., 11-dehydrothromboxane B2. The binding of the tracer is displaced by as low as 1 pg/ml of the homologous ligand, with a high degree of specificity for the open ring structure as well as for the omega side-chain. This method can detect changes in the plasma concentration and urinary excretion of 11-dehydrothromboxane B2 associated with stimulated short-term increases of thromboxane B2 secretion in the human circulation.

Increased thromboxane biosynthesis in normal pregnancy is mainly derived from platelets

Thromboxane biosynthesis was determined in normal pregnant subjects by measurement of its major urinary and plasma metabolites, 2,3-dinor-thromboxane B2 and 11-dehydro-thromboxane B2. Urinary 2,3-dinor-thromboxane B2 increased early in pregnancy (731 +/- 124 pg/mg creatinine) compared with nonpregnancy (less than 350 pg/mg creatinine; p less than 0.001) and the postpartum period (155 +/- 42 pg/mg creatinine, p = 0.015) and remained elevated throughout gestation. Similarly, plasma and urinary 11-dehydro-thromboxane B2 were increased in pregnancy. To determine the cellular origin of the increase in thromboxane biosynthesis in pregnancy, platelet cyclooxygenase was selectively inhibited with aspirin in a dose of 120 mg orally followed by 20 mg twice daily for 7 days (n = 4). Selectivity was confirmed by measurement of urinary 2,3-dinor-6-keto-prostaglandin F1 alpha, an index of prostacyclin biosynthesis. Coincident with a 97% inhibition of serum thromboxane B2, urinary 2,3-dinor-thromboxane B2 was almost completely inhibited and paralleled the recovery of platelet cyclooxygenase after withdrawal of aspirin. This study demonstrates that thromboxane biosynthesis is increased in pregnancy. The increase is mainly platelet derived and is consistent with increased platelet activation throughout pregnancy.

Measurement of renal and non-renal eicosanoid synthesis.

Enzymatic metabolites of arachidonic acid (eicosanoids) have potent biologic actions in vitro that suggest their pathophysiologic importance in vivo. To address this possibility, analytic methodology has been developed to permit study of the formation of these compounds in vivo. Both radioimmunoassay and gas chromatography-mass spectrometry have been used to measure stable but biologically inactive metabolites of the eicosanoids. Although indirect, such measures are presently the most reliable, because superfusion-bioassay lacks the specificity and precision necessary for quantitative analysis of eicosanoid formation in vivo. Measurement of eicosanoids and their hydration products and metabolites in urine represents a non-invasive approach to the assessment of eicosanoid biosynthesis. Although a tissue of origin cannot be ascribed definitely to a compound measured in urine, corroborative evidence can be obtained to indicate the predominant tissue source under physiologic and pathologic conditions. This relates particularly to the distinction between renal and extrarenal biosynthesis of these compounds. Although similar limitations apply to the measurement of eicosanoids in plasma, these may also be confounded by sources of artifact related to blood withdrawal. In the case of thromboxane B2, these concerns have been addressed by the development of methods to measure its enzymatic metabolites in plasma. Finally, formation of eicosanoids may be studied in localized compartments such as lavage or synovial fluid. Such an approach has recently provided biochemical evidence for increased formation of prostacyclin and prostaglandin E2 at the platelet-vascular interface during selective inhibition of thromboxane synthase in humans.

Measurement of thromboxane metabolites by gas chromatography-mass spectrometry.

Publisher Summary The chapter presents a study on measurement of thromboxane metabolites by gas chromatography-mass spectrometry (GC-MS). Thromboxane (Tx) A 2 is a potent vasoconstrictor and proaggregatory substance that, by virtue of these properties, may play a role in human syndromes of vascular occlusion. Aspirin (acetylsalicylic acid) that inhibits TxA 2 , has proved to be beneficial in syndromes associated with platelet activation, such as unstable angina. Measurement of 2,3-dinor-TxBz and 1 l-dehydro-TxB 2 can be accomplished by gas chromatographic (GC)-mass spectrometric (MS) techniques or by radioimmunoassays (RIA). The first step in quantitative analysis by GC-MS is to spike the samples with an internal standard. This permits accurate quantitation, irrespective of losses during purification or because of incomplete derivatization. Quantitation by GC-MS is generally performed in the selected-ion monitoring (SIM) mode by measuring the ratio of the integrated areas of the peaks corresponding to the ion for the endogenous material and the ion for the internal standard. The chapter concludes by stating that measurement of thromboxane metabolites by GC-MS provides a reliable, specific, and sensitive index of thromboxane biosynthesis. It has permitted considerable insight into the pathophysiological role of thromboxane A 2 in human disease.

The Molecular, Biochemical and Human Pharmacology of Thromboxane A2 in Renal Disease

Arachidonic acid is a constituent of the phospholipid domain of biological membranes. Activation of phospholipases results in its release into the intracellular milieu where it is subject to metabolism to biologically active compounds. In most cell types, including the platelet (1), the predominant enzyme involved in catalyzing arachidonate release is phospholipase A2 rather than phospholipase C (2). It has recently been shown that arachidonate may be subject to direct oxygenation within the cell membrane (3); the biological role of this process remains speculative.

Quantitative Analysis of Eicosanoids by Gas Chromatography-Mass Spectrometry

Eicosanoids are metabolic products derived from polyunsaturated straight-chain C 20 carboxylic acids. The most abundant substrate in humans is arachidonic acid (AA), a physiological component of the plasma membrane. Following stimulation, AA is released from an ester linkage to phospholipids and oxygenated into an array of compounds whose biological importance has been well established in vitro. On the other hand, the investigation of their potential role in human pathology depends upon assessment of their formation in vivo. Alterations in eicosanoid biosynthesis in pathological conditions and the functional consequences of their pharmacological inhibition or antagonism have indicated their pathophysiological role in vivo1–5. Specific and sensitive assays for eicosanoids have been required to address these issues.