Marie J. Stuart

A Simple Nonradioisotope Technic for the Determination of Platelet Life-Span

Acetylsalicylic acid was shown both in vivo and in vitro to prevent the platelet lipid peroxidation normally induced by the aggregating agents thrombin and epinephrine, and the sulfhydryl inhibitor N-ethylmaleimide. After aspirin ingestion, there was a significant reduction (p smaller than 0.005) in platelet lipid peroxidation, with a gradual return to base-line values over a 10-day period. With these values, a normal platelet survival curve was constructed with a mean half-life of 4.4 days (range of 2.9 to 5.9 days). These values agree with the standard 51-Cr survivals in three patients with chronic idiopathic thrombocytopenic purpura. Half-lives of 1.0, 2.5, and 4.1 days by lipid peroxide technic compared with 1.9, 2.5, and 3.9 days by the concurrent use of 51-Cr. Thus, the technic may be used to measure platelet survival.

Effects of Acetylsalicylic Acid Ingestion on Maternal and Neonatal Hemostasis

Abstract In a case–control study, we evaluated the effects of maternal ingestion of acetylsalicylic acid (aspirin) within 10 days of delivery on maternal and neonatal hemostasis. Only one of 34 control maternal-neonatal pairs (3 per cent) had hemostatic abnormalities. In 10 pairs, when maternal aspirin ingestion occurred within five days of delivery, 6 of the 10 mothers and 9 of the 10 infants had bleeding tendencies. Seven maternal-neonatal pairs in which aspirin was ingested 6 to 10 days before delivery were free of clinical bleeding. Among seven other mothers who ingested aspirin in the immediate post-partum period four of the seven (57 per cent) also had impaired hemostasis. Neonatal hemostatic abnormalities included numerous petechiae over the presenting part, hematuria, a cephalhematoma, subconjunctival hemorrhage, and bleeding from a circumcision. Maternal bleeding was confined to excessive intrapartum or post-partum blood loss. We conclude that aspirin should be avoided during pregnancy. If ingest...

Hemostatic alterations in inflammatory bowel disease - Response to therapy

Twelve patients with acute, untreated inflammatory bowel disease (IBD) were followed prospectively for coagulation and platelet function. With no symptomatic coagulopathy, abnormalities were found in all patients. With acute diseases, elevations of fibrinogen (9/12), factor V (8/12), and factor VIII (6/12) were common. Depressions of antithrombin III levels were also observed acutely (8/12). Abnormalities of platelets were both quantitative and qualitative. Thrombocytosis was present (11/12), and abnormalities in the rate and percent platelet aggregation were seen (9/10). During therapy, factors V and VIII, antithrombin III levels, and the quantitative and qualitative platelet abnormalities returned towards normal in direct correlation with sedimentation rate and clinical disease activity.

Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism.

Summary: Normal hemostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A2, measured as its stable end-product thromboxane B2 (TXB2), and vascular prostacyclin (PGI2), which inhibits platelet aggregation and is antithrombotic. Cystathionine-β-synthase deficiency is characterized by a high frequency of thromboembolic disease. We therefore studied, in vitro, the effects of homocysteine and related compounds on platelet TXB2 and vascular PGI2 formation.In paired samples of platelet rich plasma, which had been preincubated with L-homocystine (1 mM), mean production of the two platelet cyclooxygenase products, TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid increased significantly from control levels [13.6% ± 1.9 to 19.8% ± 2.1 (P < 0.02) TXB2 and 29.8% ± 4.2 to 39.4% ± 4.1 (P < 0.01) HHT]. In the presence of D.L-homocysteine (1 mM), mean platelet TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid production was also significantly increased [12.7% ± 1.5 to 16.9% ± 1.5 (P < 0.01) TXB2 and 27% ± 4 to 31% ± 4.1 (P < 0.02) HHT]. Cystine, cysteine, or methionine (1 mM) did not have similar effects in this test system. Homocysteine and homocystine were without effect on the synthesis of vascular PGI2 by umbilical artery segments [control, 0.22 ± 0.03 to 0.21 ± 0.03 ng/mg with D.L-homocysteine and 0.20 ± 0.04 control to 0.19 ± 0.04 ng/mg with D.L-homocystine]. A homocyst(e)ine-induced increase in platelet thromboxane production in the absence of an increase in vascular prostacyclin, if present in vivo, may contribute to the vascular thromboses characteristic of human homocystinemias (homocystinurias).

Iron Deficiency Anemia and Increased Urinary Norepinephrine Excretion.

Chronic iron deficiency in rats resulted in decreased MAO activity both in vitro and in vivo. Since MAO is an important enzyme in inactivation of catecholamines, urinary excretion of DA, NE, E, MN-NMN, and VMA was measured in 24-hour samples from 11 iron-deficient children before and after treatment with intramuscular iron. Pretreatment NE excretion was abnormally high and returned to normal (P=0.001) within one week of therapy. VMA excretion also was higher before than after treatment (P greater than 0.05), but most values were within the normal range for healthy children of comparable size. There was no significant difference between DA, E, and MN-NMN excretion before and after iron therapy. Anemic, non-iron-deficient children had normal urinary NE, E, and VMA excretion before and after transfusion. These findings suggest that the irritability, lack of attentiveness, and low performance scores of iron-deficient children may be related to alterations in catecholamine metabolic pathways secondary to dependence of MAO on adequate iron stores.

Vitamin E deficiency and enhanced platelet function: Reversal following E supplementation

Marked platelet hyperaggregability to adenosine diphosphate, epinephrine, and collagen was demonstrated in two children with vitamin E deficiency, with complete reversal following E supplementation. No clinical thrombotic tendency was observed during the E-deficient state. The action of vitamin E in the schema of platelet arachidonate peroxidation appears to be at the step of phosphilpase A activation, or the conversion of arachidonic acid into the cyclic endoperoxides, since the peroxidation product malonaldehyde was increased during the E-deficient state with normalization following E sufficiency.

Abnormalities of platelet aggregation in the vaso-occlusive crisis of sickle-cell anemia.

An impairment in the rate and per cent of first phase platelet aggregation with ADP was present in 10 patients with sickle-cell anemia during vaso-occlusive crises, when compared to their own base-line values and those of 20 normal control subjects. ADP released from erythrocytes or platelets could have been the mediator of this platelet “refractory state” since a concomitant decrease in platelet adhesiveness during the vaso-occlusive crises was also demonstrable. The effect of the platelet on the production or maintenance of the vaso-occlusive crisis of sickle-cell anemia requires further elucidation.

Platelet function in recipients of platelets from donors ingesting aspirin.

Abstract The effect of fresh platelet transfusions from donors ingesting aspirin was evaluated in a group of thrombocytopenic recipients. Prolonged bleeding times were corrected to normal in recipients of platelets from donors who had taken aspirin 36 hours before donation, but not in those receiving platelets from donors who had taken aspirin within 12 hours of donation. In healthy volunteers, the abnormalities induced by aspirin ingestion on the bleeding time and epinephrine-induced platelet aggregation disappeared by 72 hours after ingestion. Analysis of the data indicates that when approximately 20 per cent of the circulating platelets have not been exposed to aspirin, the hemostatic function of the platelet pool is maintained. Thus, widespread use of aspirin should not present a serious problem in the selection of platelet donors.

13-Hydroxyoctadecadienoic acid (13-HODE) stimulates prostacyclin production by endothelial cells

The effect of 13-hydroxyoctadecadienoic acid (13-HODE), an endogenous lipoxygenase metabolite of linoleic acid, on prostacyclin production by fetal bovine aortic endothelial cells was evaluated. Time-dependent release of radioimmunoassayable 6KPGF1 alpha in the presence of 13-HODE (10 uM) was stimulated by 39%, 27%, and 34% at 10, 30 and 120 min respectively. 13-HODE (10 uM) had no effect on the conversion of exogenous [14C] arachidonic acid (AA) to prostacyclin. When the effect on AA release was evaluated in [14C] AA prelabeled cells, 13-HODE (10nM) stimulated the release of AA from membrane phospholipids. Analysis of cellular phospholipids revealed a significant decrease in phosphatidylethanolamine. Our results demonstrate that 13-HODE stimulates prostacyclin production by enhancing AA release from phospholipids.

Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by cyclooxygenase

Human umbilical arteries convert arachidonic acid into three hydroxy-eicosatetraenoic acids as well as 6-ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and D2 and thromboxane B2. Two of these hydroxy derivatives of arachidonic acid were purified by reverse-phase HPLC and identified by GC-MS as 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) while a third, presumed dihydroxy derivative has not yet been identified. Both the cyclooxygenase and HETE synthesizing activities were found to be localized mainly in the microsomal fraction (100 000 X g pellet) (51 and 61% of total, respectively), and approx. 25% of both activities was found in the 10 000 X g pellet. The formation of these HETEs was inhibited by the cyclooxygenase inhibitors indomethacin and aspirin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. Production of immunoreactive 15-HETE as well as 6-ketoprostaglandin F1 alpha were also decreased significantly when arterial segments were incubated in the presence of either indomethacin or aspirin. Indomethacin inhibited the formation of both prostanoids and HETEs by microsomes in a concentration-dependent and time-dependent manner. The ID50 values for indomethacin against HETE synthesizing activity and against cyclooxygenase were 4.5 and 3.8 microM, respectively. The inactivation constants were found to be 0.09 and 0.08 min-1 for HETE synthesizing activity and cyclooxygenase, respectively. These two microsomal activities were solubilized in parallel with Tween-20. Incubation with three distinct monoclonal antibodies against different epitopes on cyclooxygenase precipitated both cyclooxygenase and HETE synthesizing activity. Each of these activities was recovered in the immune pellets. These studies demonstrate that in human umbilical arteries 11-HETE, 15-HETE and a presumed di-HETE are the products of cyclooxygenase.