Docosahexaenoic acid and docosapentanoic acid incorporation into human platelets after 24 and 72 hours: inhibitory effects on platelet reactivity

Short-term in vitro platelet membrane lipid enrichment studies and feeding trials of human subjects with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have shown a decreased reactivity in the platelet response to collagen. In this study, exogenous albumin-bound n-3 polyunsaturated fatty acids (PUFAs), namely EPA, DHA and docosapentanoic acid (DPA) were added to platelet suspensions and maintained at 22 degrees C for 24 and 72 hours. Subsequently, the aggregation response to agonist stimulation and the morphological appearance of the platelets were evaluated. A significant enrichment of platelet phospholipids (PL) in n-3 fatty acids occurred upon incubation with n-3 PUFAs in vitro, which was accompanied by a decrease in the aggregation response to collagen and preservation of platelet morphology compared with non-supplemented control platelet preparations. The inhibitory effect of the n-3 PUFAs appeared to be surface mediated in the case of DHA and DPA because the platelet response to agonist returned when the fatty acids were removed by washing. The platelet aggregation response after storage at 22 degrees C was also evaluated in platelet suspensions collected from healthy individuals before and after 42 days of dietary supplementation with seal oil, rich in DPA and DHA. Unlike the in vitro supplementation, in vivo modification and enrichment of platelet PLs by ingestion of seal oil did not appear to improve platelet function during storage relative to the placebo group.     

The acute effects of intravenous nisoldipine on left ventricular function 24 to 72 hours after uncomplicated acute myocardial infarction

Summary The acute effects on left ventricular function of nisoldipine were studied in six patients 56±12 hours (range 44 to 72 hours) after the onset of uncomplicated acute myocardial infarction. Nisoldipine was administered as a 4.5 g/kg intravenous bolus over 3 minutes followed by an infusion of 0.2 g/kg during 60 minutes. Radionuclide angiography and two-dimensional echocardiography were performed before and during infusien with nisoldipine. The left ventricular ejection fraction increased significantly from 38%±10% to 49%±10% (P=0.028) during nisoldipine infusion. Regional wall motion index was determined both by radionuclide and by two-dimensional echocardiography and showed a significant change during nisoldipine infusion from 1.9±0.3 to 1.5±0.3 (p=0.028, radionuclide angiography) and from 0.7±0.2 to 0.3±0.2 (p=0.043, two dimensional echocardiography). Heart rate increased significantly from 78±12 min^-1 to 92±13 min^-1 (p=0.028), but mean double product did not change significantly during nisoldipine infusion. It is concluded that nisoldipine significantly improves global and regional left ventricular function in patients shortly after acute myocardial infarction. This beneficial effect may, however, be partially offset by an increase in heart rate. Since mean double product did not change, it is suggested that nisoldipine may improve coronary blood flow in patients with acute myocardial infarction.     

Inhibitory effect of glyburide on thrombin-induced platelet aggregation and phosphoinositide metabolism in normal human platelets

We previously reported the effects of diet, sulphonylureas or insulin on thrombin-induced platelet aggregation, phosphoinositide metabolism and protein phosphorylation in non-insulin-dependent diabetes mellitus (NIDDM) patients. To clarify the mechanism of glyburide and insulin on platelet function, here we studied the in vitro effects of glyburide and insulin on thrombin-induced metabolic changes using normal human platelets. Platelet aggregation stimulated with <0.5 U/ml thrombin, 0.75-3 mu M adenosine diphosphate (ADP) or 1 mu g/ml collagen was significantly lower in glyburide-treated platelets, but not in insulin-treated platelets, than in untreated ones (control). Thrombin-induced incorporation of 32P radioactivity into phosphatidic acid (PA) in glyburide-treated platelets was lower than that in control but not in insulin-treated platelets. Phosphorylated proteins of platelets induced by thrombin and 12- O -tetradecanoylphorbol 13-acetate (TPA) in glyburide-treated platelets were suppressed, but not in insulin-treated platelets, compared with control. These results suggest that glyburide induces suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP2 and production of PA, and subsequently inhibits platelet aggregation.     

Inhibitory effect of glyburide on thrombin-induced platelet aggregation and phosphoinositide metabolism in normal human platelets

We previously reported the effects of diet, sulphonylureas or insulin on thrombin-induced platelet aggregation, phosphoinositide metabolism and protein phosphorylation in non-insulin-dependent diabetes mellitus (NIDDM) patients. To clarify the mechanism of glyburide and insulin on platelet function, here we studied the in vitro effects of glyburide and insulin on thrombin-induced metabolic changes using normal human platelets. Platelet aggregation stimulated with <0.5 U/ml thrombin, 0.75-3 microM adenosine diphosphate (ADP) or 1 microg/ml collagen was significantly lower in glyburide-treated platelets, but not in insulin-treated platelets, than in untreated ones (control). Thrombin-induced incorporation of 32P radioactivity into phosphatidic acid (PA) in glyburide-treated platelets was lower than that in control but not in insulin-treated platelets. Phosphorylated proteins of platelets induced by thrombin and 12- O -tetradecanoylphorbol 13-acetate (TPA) in glyburide-treated platelets were suppressed, but not in insulin-treated platelets, compared with control. These results suggest that glyburide induces suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP(2) and production of PA, and subsequently inhibits platelet aggregation.     

Inhibitory effects of Cyperus digitatus extract on human platelet function in vitro

The purpose of this research was to investigate the mechanisms of antiplatelet action of Cyperus digitatus. The antiplatelet action of C. digitatus was studied on platelet function: secretion, adhesion, aggregation, and sCD40L release. The platelet ATP secretion and aggregation were significantly inhibited by CDA (ethyl acetate extract) at 0.1?mg/ml and after the incubation of whole blood with CDA, the platelet coverage was inhibited by 96?±?3% (p?<?0.001). At the same concentration, CDA significantly decreased sCD40L levels. The mechanism of antiplatelet action of CDA could be by NF-κB inhibition and that is cAMP independent. In conclusion, C. digitatus extract may serve as a new source of antiplatelet agents for food and nutraceutical applications.     

Inhibitory effects of staphylococcal enterotoxin type B on human platelet adhesion in vitro

Septic shock was formerly recognized as a consequence of Gram-negative bacteraemia, but at present the incidence of Gram-positive sepsis seems to be more relevant, contributing for more than 50% of cases. Staphylococcal aureus can induce toxic shock in humans through the production of potent toxins termed Staphylococcal enterotoxins, from which Staphylococcal enterotoxin type B (SEB) is one of most studied. Platelets are reported to participate in pathogenesis of severe sepsis, but the exact role of platelets in this event is poorly investigated, particularly that caused by Gram-positive bacteria. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the actions of SEB on human platelets. Ninety-six-well microtiter plates were coated with human fibrinogen (50 µg/mL), and human washed platelet suspension (6 × 10⁶ platelets) was added to each well. Adherent platelets were quantified through measurement of acid phosphatase activity. Staphylococcal enterotoxin B (0.0001–30 µg/mL, incubated for 5 to 60 min) time- and dose-dependently inhibited platelet adhesion. This response was modified neither by the protein synthesis inhibitor puromycin (0.01 and 0.1 mM) nor by the superoxide scavengers superoxide dismutase (SOD, 100 units/mL) and polyethylene glycol-SOD (30 U/mL). The peroxide hydrogen (H₂O₂) scavenger catalase polyethylene glycol (1000 U/mL) significantly attenuated the platelet adhesion inhibition by SEB. The cAMP and cGMP levels were not changed by SEB (0.0001–30 µg/mL, 60 min). Our findings suggest that H₂O₂ at least partly contributes to the inhibitory responses of human platelet adhesion by SEB.     

An investigation into the effects of bacterial lipopolysaccharide on human platelets

The addition of lipopolysaccharide (LPS) from Gram-negative bacteria to whole blood induced a consistent enhancement of platelet impedance aggregation (WB-PIA) and an increase in median platelet volume (MPV). In contrast, the addition of LPS to platelet rich plasma (PRP) resulted in inconsistent changes in turbidometric platelet aggregation (TPA) and no significant change in MPV. The LPS-induced increase of MPV in whole blood was inhibited by verapamil, a calcium channel blocker. We conclude that: (a) LPS consistently activates platelets in whole blood but not in PRP; (b) LPS-induced activation of platelets is probably mediated by products released by leucocytes and/or erythrocytes; (c) the increase in MPV induced by LPS is probably dependent upon calcium influx; and (4) an increase in MPV may be a useful and sensitive model for the study of platelet activation by LPS, in vitro and in vivo.     

Inhibitory effects of staphylococcal enterotoxin type B on human platelet adhesion in vitro

Septic shock was formerly recognized as a consequence of Gram-negative bacteraemia, but at present the incidence of Gram-positive sepsis seems to be more relevant, contributing for more than 50% of cases. Staphylococcal aureus can induce toxic shock in humans through the production of potent toxins termed Staphylococcal enterotoxins, from which Staphylococcal enterotoxin type B (SEB) is one of most studied. Platelets are reported to participate in pathogenesis of severe sepsis, but the exact role of platelets in this event is poorly investigated, particularly that caused by Gram-positive bacteria. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the actions of SEB on human platelets. Ninety-six-well microtiter plates were coated with human fibrinogen (50 microg/mL), and human washed platelet suspension (6 x 10(6) platelets) was added to each well. Adherent platelets were quantified through measurement of acid phosphatase activity. Staphylococcal enterotoxin B (0.0001-30 microg/mL, incubated for 5 to 60 min) time- and dose-dependently inhibited platelet adhesion. This response was modified neither by the protein synthesis inhibitor puromycin (0.01 and 0.1 mM) nor by the superoxide scavengers superoxide dismutase (SOD, 100 units/mL) and polyethylene glycol-SOD (30 U/mL). The peroxide hydrogen (H(2)O(2)) scavenger catalase polyethylene glycol (1000 U/mL) significantly attenuated the platelet adhesion inhibition by SEB. The cAMP and cGMP levels were not changed by SEB (0.0001-30 microg/mL, 60 min). Our findings suggest that H(2)O(2) at least partly contributes to the inhibitory responses of human platelet adhesion by SEB.     

123I thyroid uptake and thyroid size at 24, 48, and 72 hours after the administration of recombinant human thyroid-stimulating hormone to normal volunteers

CONTEXT: Recombinant human TSH (rhTSH) is used to evaluate thyroid carcinoma patients and off-label for (131)I thyroid ablation and nontoxic goiter therapy. OBJECTIVE: Our objective was to determine the optimal time for (131)I administration after rhTSH. PARTICIPANTS: Twenty-five euthyroid nongoitrous volunteers participated in the study. DESIGN: Baseline 24-h thyroid (123)I uptake (RAIU) was measured, and then 0.1 mg rhTSH was administered. (123)I was administered 24, 48, or 72 h after rhTSH, and a repeat 24-h RAIU was obtained. SETTING: The study was conducted at an academic research center. MAIN OUTCOME MEASURES: Thyroid function tests, thyroid ultrasounds, and electrocardiograms were measured before rhTSH, then daily for 4 d, and finally 7 d after rhTSH. RESULTS: Serum TSH concentrations 24 h after rhTSH increased from 1.7 +/- 0.5 muU/ml (mean +/- sd) to 13.3 +/- 4. The 24-h RAIUs rose from 25 +/- 5 to 47 +/- 8% (88% increase) when the (123)I was given at 24 h after rhTSH and from 29.8 +/- 7 to 40.5 +/- 13% (36% increase) when the (123)I was given at 48 h and were unchanged when the (123)I was given at 72 h. The post-rhTSH RAIU increase was greater at 24 than at 72 h (P < 0.005) and marginally greater than at 48 h (P = 0.057). Thyroid volumes significantly increased 48 h after rhTSH (10 +/- 3.8 vs. 11.1 +/- 3.7 ml; P < 0.009). Electrocardiograms were normal. CONCLUSIONS: Marked increases in RAIU occurred when (123)I was given 24 h after rhTSH administration to euthyroid volunteers. Smaller increases were observed at 48 h and none at 72 h.     

Total sialic acid in human and canine platelets does not change with the platelet age.

Total platelet sialic acid (SA) was measured in three experimental conditions: (1) human and canine platelet density subpopulations obtained by centrifugation in arabinogalactan gradients, (2) circulating canine platelets during recovery from experimental immune and mechanical thrombocytopenias, and (3) platelets obtained from a patient with chronic immune thrombocytopenic purpura before and after splenectomy. The density of human and canine platelets is, in part, determined by their age. We found no significant differences in total SA between high-density (HD) and low-density (LD) platelets (9.32 +/- 2.0 vs. 9.55 +/- 1.3 micrograms/mg of platelet protein in dogs and 9.02 +/- 2.3 vs. 9.10 +/- 2.9 micrograms/mg in humans). In the human and canine thrombocytopenic models, the entrance of new platelets from the bone marrow is followed by their aging in the circulation. In these models, no significant changes in total SA content were detected in sequential measurements during the recovery of the thrombocytopenia. Accordingly, we conclude that total SA in human and canine platelets is unrelated to their age in circulation. These results do not support the notion that the loss of SA from membrane glycoproteins determines the recognition and removal of platelets from the circulation.     

Multiple active forms of thrombin: binding to platelets and effects on platelet function.

The effect of various forms of thrombin on certain platelet functions has been investigated. Partially purified bovine thrombin which is a mixture of multiple active forms of thrombin, was chromatographed to yield molecular species termed alpha-, beta-, and gamma-thrombin, each of which has varying degrees of fibrinogen clotting and esterase activities. A direct correlation was observed between the ability of the different forms of thrombin to clot fibrinogen and to influence platelet function. In general, thrombin with high fibrinogen clotting activity was also a potent inducer of platelet aggregation and the release reaction, while those species with low clotting ability were poor inducers of aggregation and release.     

Inhibitory effects of endothelial cells and calcium channel blockers on platelet aggregation

This study examined the effects of human umbilical vein endothelial cells (ECs) and calcium channel blockers (Ca2+ blockers), diltiazem, verapamil, and nicardipine, on platelet aggregation in vitro. ECs markedly inhibited the platelet aggregation induced by ADP, collagen, thromboxane A2 (TXA2), or thrombin. When platelets were incubated with ECs, the antiaggregatory activity reached a plateau within 5 to 10 min. As the number of ECs added to the platelet-rich plasma was increased, platelet aggregation declined progressively. The antiaggregatory activity of ECs was attenuated considerably by the addition of aspirin. All three Ca2+ blockers inhibited platelet aggregation in a dose-dependent manner. The combination of ECs and a Ca2+ blocker resulted in more potent inhibition of platelet aggregation than either alone, but the effects were not synergistic. Both ECs and Ca2+ blockers inhibited the synthesis of TXA2 during platelet aggregation. However, Ca2+ blockers did not significantly influence the production of prostaglandin I2 (PGI2) by ECs during incubation and aggregation. These results suggest that PGI2 is an important factor in endothelial antiaggregatory activity. Ca2+ blockers directly inhibit platelet aggregation by suppressing TXA2 formation, but do not appear to enhance the antiaggregatory activity of ECs.