Higher platelet reactivity and platelet-monocyte complex formation in Gram-positive sepsis compared to Gram-negative sepsis.

Platelets may play a role in the high risk for vascular complications in Gram-positive sepsis. We compared the platelet reactivity of 15 patients with Gram-positive sepsis, 17 with Gram-negative sepsis and 20 healthy controls using a whole blood flow cytometry-based assay. Patients with Gram-positive sepsis had the highest median fluorescence intensity (MFI) of the platelet membrane expression of P-selectin upon stimulation with high dose adenosine diphosphate (ADP; P = 0.002 vs. Gram-negative and P = 0.005 vs. control groups) and cross-linked collagen-related peptide (CRP-XL; P = 0.02 vs. Gram-negative and P = 0.0001 vs. control groups). The Gram-positive group also demonstrated significantly higher ADP-induced fibrinogen binding (P = 0.001), as wll as platelet-monocyte complex formation (P = 0.02), compared to the Gram-negative group and had the highest plasma levels of platelet factor 4, ß-thromboglobulin and soluble P-selectin. In contrast, thrombin-antithrombin complex and C-reactive protein levels were comparable in both patient groups. In conclusion, common Gram-positive pathogens induce platelet hyperreactivity, which may contribute to a higher risk for vascular complications.

Antithrombotic triple therapy and coagulation activation at the site of thrombus formation: a randomized trial in healthy subjects.

BACKGROUND: Patients with acute coronary syndrome and concomitant atrial fibrillation may require antithrombotic triple therapy but clinical evidence of safety and efficacy is poor. We have therefore studied the combination of different antithrombotic medicines for coagulation activation in an in vivo model in the skin microvasculature. METHODS AND RESULTS: Platelet activation (ß-thromboglobulin [ß-TG]) and thrombin generation (prothrombin fragment 1 + 2 [F1+2 ], thrombin-antithrombin complex [TAT]) were studied in an open-label, randomized, parallel group trial in 60 healthy male subjects (n = 20 per group) who received ticagrelor and acetylsalicylic acid (ASA) in combination with dabigatran (150 mg bid), rivaroxaban (20 mg od) or phenprocoumon (INR 2.0-3.0). Coagulation biomarkers in shed blood were assessed at 3 h after monotherapy with the medicines under study, at 3 h after triple therapy dosing and at steady state trough conditions. Single doses of ticagrelor, dabigatran or rivaroxaban caused comparable decreases in shed blood ß-TG and were more pronounced than phenprocoumon at an INR of 2.0-3.0. In contrast, thrombin generation was more affected by rivaroxaban and phenprocoumon than by dabigatran. During triple therapy a similarly sustained inhibition of platelet activation and thrombin generation with a maximum decrease of ß-TG, F1+2 and TAT at 3 h post-dosing was noted, which remained below pre-dose levels at trough steady state. CONCLUSION: A triple therapy at steady state with ticagrelor plus ASA in combination with dabigatran or rivaroxaban is as effective as a combination with phenprocoumon for platelet activation and thrombin generation in vivo.

Inhibition of PAR4 signaling mediates ethanol-induced attenuation of platelet function in vitro.

BACKGROUND: Reduction in coronary heart disease morbidity in response to moderate consumption of alcoholic beverages may be partly mediated by ethanol-induced inhibition of platelet function. However, the precise mechanisms by which ethanol modulates platelet activation induced by thrombin, which plays a central role in hemostasis, remain unclear. The goal of this study was to investigate ethanol-induced changes in platelet function and clarify the underlying mechanisms including PAR1 and PAR4 activity and [Ca2+]i dynamics in vitro. METHODS: Platelet aggregation, increase in intracellular calcium ([Ca2+]i), and release of platelet factor 4 and beta-thromboglobulin induced by alpha-thrombin, PAR1-agonist peptide (AP), or PAR4-AP were assessed in the presence or absence of ethanol. RESULTS: Ethanol exposure inhibited low-dose thrombin (0.5 nM)-induced aggregation but not an increase in [Ca2+]i. In contrast, ethanol had no effect on high-dose thrombin (10 nM)-induced aggregation or the [Ca2+]i increase. Ethanol did not significantly inhibit thrombin-induced release of platelet factor 4 and beta-thromboglobulin. Ethanol reduced PAR1-AP-induced aggregation, but did not affect the spike form of [Ca2+]i increase. In contrast, ethanol inhibited the increase in [Ca2+]i as well as the aggregation in response to PAR4-AP and resulted in delayed [Ca2+]i peak time. Furthermore, ethanol inhibited both PAR1-AP- and PAR4-AP-induced platelet factor 4 and beta-thromboglobulin release. CONCLUSIONS: These data suggest that ethanol inhibits platelet aggregation via inhibition of PAR4 signaling and subsequent inhibition of Ca2+ influx and granule release. This phenomenon may contribute to the reduction in coronary heart disease morbidity in response to consumption of alcoholic beverages.

Transvenous cryoablation reduces platelet activation during pulmonary vein ablation compared with radiofrequency energy in patients with atrial fibrillation.

BACKGROUND: Radiofrequency (RF) ablation procedures for atrial fibrillation (AF) are associated with potential risks of thromboembolism, which may be minimized by the use of cryoablation that preserves the integrity of endocardium. The objective of this study was to compare the thrombogenic potential of transvenous cryoablation versus RF ablation during pulmonary vein (PV) isolation. METHODS AND RESULTS: Thirty consecutive patients with paroxysmal AF were randomized to undergo segmental PV isolation procedure using 4-mm tip RF ablation (n = 15) or cryoablation (CryoCor, San Diego, CA, USA) (n = 15). Blood samples were drawn after sheath insertion (baseline), after transseptal puncture, before ablation (after heparin administration), and after isolation of a superior PV. Activation of coagulation was measured with plasma levels of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin III complex (TAT), and platelets by plasma level of beta-thromboglobulin (beta-TG) and flow cytometric enumerating of P-selectin (CD62)-positive platelets. In both groups, the plasma level of beta-TG, F1 + 2, and TAT were elevated after sheath insertion. The percentage changes in plasma level of beta-TG, F1 + 2, and TAT and CD41/62-positive platelets from baseline after transseptal puncture and before ablation were similar (P > 0.05). However, the percentage changes in CD62-positive platelets from baseline were significantly higher in patients treated with RF ablation (82 +/- 20%) than with cryoablation (22 +/- 14%, P = 0.02), although their plasma levels of beta-TG, F1 + 2, and TAT were not different (P > 0.05). CONCLUSIONS: Significant platelet and coagulation activations were observed during PV ablation procedures, and heparin administration only prevented activation of coagulation but not platelets. Persistent platelets activation was observed during RF energy application, but not during cryoablation.

Storage of platelets in additive solutions: the effects of magnesium and potassium on the release of RANTES, beta-thromboglobulin, platelet factor 4 and interleukin-7, during storage.

BACKGROUND AND OBJECTIVES: Several studies have suggested that the accumulation of cytokines during storage of platelet concentrates may mediate non-haemolytic transfusion reactions. Prestorage leucodepletion can prevent the release of cytokines from white blood cells during storage, but not the release of platelet-derived cytokines. Therefore, we investigated whether the addition of magnesium and potassium to platelets stored in a platelet additive solution (PAS) would affect the generation of cytokines during platelet storage. MATERIALS AND METHODS: Platelets were prepared from buffy coats using different suspension media: plasma; 70% PAS-III + 30% plasma; 70% PAS-III supplemented with magnesium and potassium +30% plasma; and 80% PAS-III supplemented with magnesium and potassium +20% plasma. The levels of certain cytokines--regulated on activation, normal, T-cell expressed, and secreted (RANTES), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and interleukin-7 (IL-7)--were measured by enzyme-linked immunosorbent assay (ELISA) on days 1, 5 and 7. RESULTS: The concentrations of RANTES, beta-TG, PF4 and IL-7 increased, during storage, in all units. The increase was significantly greater in units stored in 70% PAS-III +30% plasma than in the other three suspension media. The storage of platelets in 70% PAS-III supplemented with magnesium and potassium +30% plasma significantly reduced the concentrations of platelet derived-cytokines during storage, as compared to platelets stored in 70% PAS-III + 30% plasma alone. CONCLUSIONS: The concentrations of platelet-derived cytokines increased, to a significantly greater extent, when platelets were stored in PAS-III than in plasma. However, when magnesium and potassium were added to PAS-III, the concentrations of platelet-derived cytokines obtained during storage were about the same as those produced by platelets stored in plasma.

Moderate supplementation with natural alpha-tocopherol decreases platelet aggregation and low-density lipoprotein oxidation.

Previous studies have shown that oral administration of 300 mg alpha-tocopherol/day to healthy volunteers decreases platelet function and enhances their sensitivity to the platelet inhibitor, prostaglandin E(1), when full dose-response curves to a range of agonist concentrations are made. In this study, the effects of oral doses of natural alpha-tocopherol (75, 200 and 400 IU/day) were studied in order to determine whether the same effects might be achieved with lower intakes of vitamin E and whether inhibition is related to the platelet levels of the antioxidant in platelet membranes. Twenty two subjects undertook the supplementation regime, divided into three units of 2 weeks, each cycling through each of the dosages. The results show that uptake of vitamin E by the platelets was optimal at 75 IU/day, correlating with the maximal influence on platelet aggregation and platelet responsiveness to inhibition by PGE1, increased supplemental levels exerting no greater effects.

The influence of antioxidant nutrients on platelet function in healthy volunteers.

There is mounting evidence that antioxidants may help to prevent coronary heart disease and modulate some thrombotic events such a platelet adhesion. However, the effects of antioxidant supplementation on platelet function in vivo are controversial. A double-blind, randomised, placebo-controlled study was performed on 40 healthy volunteers (20-50 years) supplemented daily with vitamin E (300 mg), vitamin C (250 mg) or beta-carotene (15 mg) for 8 weeks. Platelet function was assessed by platelet aggregation induced by ADP, arachidonic acid or collagen, platelet responsiveness to the inhibitor PGE1, beta-thromboglobulin release and ATP secretion. Supplementation with vitamin E resulted in a significant increase in platelet alpha-tocopherol level (+68%) reflecting closely the increase in plasma alpha-tocopherol level (+69%). Platelet function was significantly decreased by vitamin E as revealed by the decreased platelet aggregation in response to ADP and arachidonic acid, the increased sensitivity to inhibition by PGE1, the decreased plasma beta-thromboglobulin concentration and the decreased ATP secretion. Supplementation with vitamin C did not affect platelet function significantly although a trend towards a decreased platelet aggregability and an increased sensitivity to the inhibitor PGE1 were observed. No significant changes in platelet function occurred after supplementation with beta-carotene. In conclusion, supplementation of healthy volunteers with vitamin E decreased platelet function whereas supplementation with vitamin C or beta-carotene had no significant effects.

Activation of circulating platelets by hyperthermal stress.

To determine the involvement of activated platelets in the frequent thrombosis after hot hot-spring bathing, we examined effects of a hyperthermal stress on platelets in vivo. Plasma levels of beta-thromboglobulin (beta-TG) began to rise at 5 minutes and elevated significantly 10 minutes after the start of traditional 47 degrees C hot-spring bathing. In contrast, there was no significant factor 4 change in plasma beta-TG level through a control 10-minute bath in the same water at 42 degrees C. A beta-TG to platelet factor 4 ratio, a value suggested to be useful for evaluating clinical studies for in vitro versus in vivo granule release, was sufficiently high in each sampling, indicating that the experiment was performed without being influenced by artificial platelet activation. These data show that only a few minutes' hyperthermal stress can induce granule release from platelets in vivo.

Effects of a dried garlic preparation on fibrinolysis and platelet aggregation in healthy subjects.

The acute and chronic effects of a preparation of dried garlic powder (Sapec) in a total daily dose of 900 mg on fibrinolysis and platelet aggregation have been evaluated in a randomized, double-blind, placebo controlled cross-over study of 12 healthy subjects. Total euglobulin fibrinolytic activity and t-PA (tissue plasminogen activator) activity were significantly higher 4 and 6 h after garlic and placebo ingestion, and no differences were recorded between treatments. After 14 days of treatment, t-PA activity was significantly higher after garlic, with inter-treatment significance. No significant changes in PAI (Plasminogen Activator Inhibitor) activity and fibrinogen levels were recorded. Platelet aggregation induced by adenosine diphosphate and collagen, and especially beta-thromboglobulin (beta-TG) release after collagen stimulation were significantly inhibited 2 and 4 h after garlic ingestion; platelet aggregation values were also significantly lower after 7 and 14 days of garlic treatment. No significant changes were found in adenosine triphosphate release and serum TXB2 levels after acute garlic administration.

Significance of platelet beta-adrenoceptors for platelet responses in vivo and in vitro.

The significance of platelet beta-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the beta-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed alpha- and beta-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma beta-thromboglobulin levels. Adrenaline levels were 3-4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels. beta-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that beta-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an alpha-adrenoceptor mediated proaggregatory action.

[Simultaneous detection of the platelet aggregation and release in patients with cardio-cerebral vascular diseases associated with blood stasis and their clinical significance].

The authors determined the platelet aggregation(PA) activity respectively with electric impedance and photoelectric turbidimetry in patients with ischemic cardio-cerebral vascular diseases associated with blood stasis. The results showed the PA activities were elevated both in whole blood and plasma. Then, the authors detected simultaneously the PA activity and amount of post-aggregation beta-thromboglobulin(beta-TG) releasing and also in vivo amount of spontaneous plasma releasing of beta-TG with photoelectric turbidimetry and RIA methods with blood stasis. The results showed, during the acute phase of stroke, a high activated state of platelet existed, expressed as significant elevation both of the amount of beta-TG releasing of post-aggregation and plasma beta-TG level. However, no definite correlation between rate of PA and subsequent amount of beta-TG releasing was found, and detection of aggregation rate alone did not disclose the state of activation. As compared with the acute phase, during the recovery stage of stroke in which the clinical symptom of blood stasis was improved, the plasma beta-TG level declined significantly, however, was still higher than in normal controls; amount of releasing beta-TG was declining which denoted that the platelet functions were reducing then, but were still in a higher state of activation. These results suggested that there were changes both in number and quality of platelet in patients with blood stasis.

[Effects of Ligusticum wallichii on the plasma levels of beta-thromboglobulin, platelet factor 4, thromboxane B2 and 6-keto-PGF1 alpha in rabbits under acute experimental cerebral ischemia].

By occluding the bilateral carotid arteries of rabbits to produce bilateral partial cerebral ischemia, and by using RIA and ELISA to measure the levels of Beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in plasma, the authors found that the levels of beta-TG, PF4 and TXB2 in plasma had significantly increased (P less than 0.01), but the level of 6-keto-PGF1 alpha in plasma showed no change (P greater than 0.05) after cerebral ischemia appeared. The results of the Ligusticum wallichii (Ligusticum) pre-treatment to the test-group showed that the levels of beta-TG, PF4 and TXB2 in plasma had significantly decreased (P less than 0.01), and the level of 6-keto-PGF1 alpha in plasma had significantly increased (P less than 0.05). This suggested that the Ligusticum treatment could effectively inhibit the platelet activation in vivo and correct the TXA2-PGI2 imbalance in blood after cerebral ischemia. In this study, some new approaches were explored to explain the mechanisms of Ligusticum for preventing and treating cerebral ischemia.

[Effect of the platelet-activating factor antagonist BN 52063 on exertional asthma]. / Wirkung des PAF-Antagonisten BN 52063 auf Anstrengungs-Asthma.

In a randomised single-blind crossover study we assessed the effects of a specific PAF acether antagonist, BN 52063, on the early asthmatic response to exercise in six patients with exercise induced asthma. After a treatment period of two days an exercise challenge on the third day was preceded by administration of either placebo or BN 52063 240 mg p.o. 3 hours or 5 mg by inhalation 30 minutes before the challenge. After the oral intake of 240 mg BN 52063 there was no effect on the initial exercise induced bronchoconstriction, but the prolonged reduction of PEF was significantly attenuated expressed as a smaller AUC (p less than 0.02). In the placebo period there was a marked increase in plasma concentrations of both platelet factor 4 (PF4) and beta-thromboglobulin (beta-TBG). Intake of BN 52063 diminished the rise in plasma concentrations of PF4 and beta-TBG after the exercise challenge significantly. The results show that platelet activation after exercise induced asthma was markedly inhibited by BN 52063, indicating that PAF acts as a mediator in exercise induced asthma.

Ajoene, the antiplatelet compound derived from garlic, specifically inhibits platelet release reaction by affecting the plasma membrane internal microviscosity.

Ajoene (E,Z-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a product of the rearrangement of allicin (a major component of raw garlic), has been shown to be a potent inhibitor of platelet aggregation in vitro through inhibition of granule release and fibrinogen binding. Our present study further elaborates on this inhibitory action, through studies of the effect of ajoene on the earliest steps of platelet activation. The transducing mechanism involved in thrombin-induced platelet activation was not modified by the drug as indicated by a normal breakdown of phosphatidylinositol 4,5,bisphosphate and normal production of phosphatidic acid. Likewise, the agonist-induced phosphorylation of myosin light chain (P20) and of the 43 kD protein (P43) were not impaired by ajoene. Under the same conditions, however, ajoene (100 microM) produced a strong inhibition of the thrombin-induced release of dense body and alpha-granule constituents. Electron spin resonance studies of the effect of ajoene on some physico-chemical properties of the platelet plasma membrane (intact platelets), as well as on artificial lipid membranes, indicated that ajoene increased mobility of the fatty acid spin label 16 nitroxide stearate. This suggests the existence of a decreased microviscosity of the most internal region within the lipid bilayer membrane, without affecting the outer hydrophilic moieties of the bilayer. As a whole, these results suggest that the effect of ajoene on the release reaction must be, in part, due to physical modification of the bilayer, which impairs the fusion of the granules and plasma membrane, a prerequisite for exocytosis.

Selenium supplementation does not alter platelet activation in subjects with normal selenium status.

The effect of Se supplementation on the plasma concentrations of platelet specific proteins, beta-thromboglobulin (beta TG) and platelet factor 4 (PF4), was determined in twenty young women with normal selenium (Se) status using a double blind protocol. Selenium supplementation for 4 weeks (150 micrograms/day), did not elevate the initial mean plasma Se level 95 +/- 4 ng/ml above this level, nor did it alter the plasma beta TG/PF4. Moreover, all the other parameters of the body antioxidative status (plasma alpha-tocopherol, retinol and uric acid and whole blood glutathione) measured in this experiment stayed unaltered during the 4-week supplementation period. The results indicate no relationship between Se supplementation and platelet function in subjects with normal Se status.

Lack of efficacy of nafazatrom, a novel anti-thrombotic compound, in patients with coronary artery disease.

Nafazatrom (Bay G 6575) is a novel antithrombotic compound, which acts by stimulation of prostacyclin as well as by inhibition of lipoxygenase enzymes. To determine its effects on exercise performance in coronary artery disease patients, a double-blind study was conducted. Twenty patients with coronary artery disease underwent an exercise stress test before and 2 hours after administration of placebo or nafazatrom (1.2 gm). Before the drug administration, there was evidence of enhanced platelet activity, as reflected by elevated resting plasma beta thromboglobulin and thromboxane B2 concentrations. Plasma 6-keto-PGF1 alpha levels were undetectable in most patients. All coagulation tests were in the normal range. None of these parameters changed with exercise. Administration of placebo or nafazatrom before the exercise stress test did not significantly influence any of the coagulation or platelet function parameters or plasma concentrations of thromboxane B2 and 6-keto-PGF1 alpha. This lack of effect was evident both at rest and upon exercise. Compared to placebo, nafazatrom did not significantly increase exercise tolerance time or exercise-induced symptoms. In conclusion, nafazatrom did not influence exercise performance in patients with coronary disease.

Eosinophil cationic protein in inflammatory synovial effusions as evidence of eosinophil involvement.

Eosinophils are seldom noted in inflammatory synovial fluids but are reported to infiltrate the synovial tissue in inflammatory arthritides. To elucidate a possible role for eosinophils in inflammatory joint reactions the concentrations of eosinophil cationic protein (ECP)--a specific granule protein from eosinophils--were measured by radioimmunoassay in 90 synovial fluids from patients with various inflammatory arthritides (rheumatoid arthritis, reactive and crystal arthritides, Reiter's disease and psoriatic arthropathy). In the same specimens lactoferrin was measured as an indicator of neutrophil-involved inflammation. In comparison with the normal circulating levels of ECP and lactoferrin the measured synovial fluid concentrations of both proteins were considerably raised in all patient groups with inflammatory joint diseases in contrast to patients with non-inflammatory arthritides. There was a striking positive correlation between the ECP and lactoferrin synovial fluid concentrations. These data indicate that eosinophil activation is prominent in inflammatory joint reactions and is linked to the activation of neutrophils. The regulation of degranulation or secretion by eosinophils is unknown. Our in-vitro studies showed that peripheral blood isolated neutrophils as well as eosinophils degranulated when exposed to IgG complexes. However, eosinophil degranulation was modest compared with neutrophil degranulation. These data suggest that neutrophil phagocytosis of, for example, immune complexes may be one major mechanism in neutrophil degranulation but that other factors determine the appearance of eosinophil products in inflammatory synovial effusions. The possible modulatory or harmful role of eosinophils in inflammatory joint disease can at present only be speculated on.