Platelet Adenosine Triphosphate Research Articles

Early predictive value of platelet function for clinical outcome in sepsis

Sepsis is the leading course of morbidity and mortality in critically ill patients. This study aimed to evaluate the predictive value of the platelet aggregation for mortality in patients with sepsis. In addition, the relationship between impaired mitochondria and the platelet aggregation was explored. This was a prospective, observational cohort study. The platelet aggregation rate in response to adenosine diphosphate (ADP) was assessed. The primary outcome was 28-day mortality. Platelet mitochondrial parameters, including adenosine triphosphate(ATP), mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening, were measured. Platelet mitochondrial ultrastructure was observed using transmission electron microscopy. 86 patients with 65 survivors and 21 non-survivors were enrolled. Platelets of non-survivors with sepsis were hyporesponsive to ADP, in terms of maximal aggregation rate (P<0.001). Receiver operating characteristic curves analysis demonstrated that the AUC estimated 28-day mortality for platelet aggregation rate was 0.814. At the optimal cut-off value of 35.8% for platelet aggregation rate, the sensitivity was 86.2% and the specificity was 66.7%. Kaplan-Meier analysis showed that a platelet aggregation rate of less than 35.8% was associated closely with poor survival. After adjusting for lactate by Cox regression analysis, platelet aggregation rate was identified as an independent predictor for the probability of 28-day mortality. Compared with survivors, non-survivors showed a significant reduction in platelet ATP and MMP-index (both P<0.001), and a remarkable increase in mPTP opening (P<0.001). ATP and MMP-index were positively correlated with platelet aggregation rate (R square=0.75, R square=0.44, respectively). Platelet aggregation rate could be an early predictive biomarker for mortality in sepsis. Impaired platelet mitochondrial activity affects platelet aggregation and correlates with the severity of sepsis.

Purine Metabolism in Platelets and Heart Cells of Hyperlipidemic Rats.

Hyperlipidemia, characterized by an increase in circulating lipid levels, doubles the chance of developing cardiovascular diseases. It prompts inflammation, immune activation, and oxidative stress in the bloodstream and organs of rats. Thus, we theorized that the metabolism of purines, an immunomodulatory mechanism, is altered in cells involved in the development of cardiovascular diseases. Therefore, we induced acute hyperlipidemia in Wistar rats with Poloxamer-407 and euthanized the animals 36h later. The leucocyte differential, the rate of purine metabolism on the surface of platelets and heart cells, and markers of oxidative stress in the heart tissue were evaluated. These parameters were also assessed in animals pretreated for 30days with curcumin and/or rutin. Hyperlipidemia increased the hydrolyses of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in platelets. In heart cells, the metabolism of ATP and adenosine (ADO) were increased, while ADP hydrolysis was reduced. Additionally, lipid damage and antioxidant defenses were increased in heart homogenates. Hyperlipidemic rats alsoexhibited a reduced percentage of eosinophils and lymphocytes. Together, these findings are indicative of an increased risk of developing cardiovascular diseases in hyperlipidemic rats. The pretreatments with antioxidants reverted some of the changes prompted by hyperlipidemia preventing detrimental changes in the cells and tissues. Graphical Abstract.

Update on diagnostic testing for platelet function disorders: What is practical and useful?

Platelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis. This review summarizes the authors' experiences, current literature, and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD. Blood counts, blood film review, and aggregation tests are the most commonly performed investigations for PFD and help determine whether there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these tests detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release show greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high-quality evidence, limit their ability to offer an expanded test menu for PFD. PFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.

Platelet storage in more than 90% additive solution containing glucose and bicarbonate has the potential to increase shelf life.

Platelet concentrates (PCs) suspended in more than 90% additive solution (AS) are recommended for patients with reactions to platelets stored in plasma. Next-generation AS containing glucose and bicarbonate might enable storage of these PCs for longer than those in current-generation AS, which was therefore evaluated in this study. Five buffy coat or apheresis-derived PCs were pooled and split into identical units. All units except the control were centrifuged, the plasma removed and replaced with AS (SSP+, PAS-5, or PAS-G), or resuspended in the same plasma and sampled 96 hours after resuspension for analysis. Plasma carryover was less than 10%, total protein less than 1 g/unit, and immunoglobulin A levels lower than 0.1 mg/mL for all PCs in AS. The pH of all the platelets during storage was higher than 6.4. PAS containing glucose maintained superior in vitro platelet function during storage compared with those resuspended in SSP+. Compared with storage in SSP+, storage in PAS-5 or PAS-G resulted in better preservation of platelet adenosine triphosphate and hypotonic shock response, lower annexin V binding, and improved mitochondrial membrane potential. Platelets resuspended in PAS-5 and PAS-G maintained in vitro function and metabolism during storage compared with SSP+ platelets. Elevated platelet metabolic activity was noticed in PAS-G, and higher platelet activation was detected with PAS-5. Platelets resuspended in PAS containing glucose has the potential to increase the shelf life of PC in more than 90% AS.

Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It?

Objective:The aim of this study was to investigate the effects of imatinib mesylate on platelet aggregation and adenosine triphosphate (ATP) release in chronic myeloid leukemia patients.Materials and Methods:Platelet aggregation and ATP release induced by 5.0 mM adenosine diphosphate, 0.5 mM arachidonic acid, 1.0 mg/mL ristocetin, and 2 µg/mL collagen were studied by whole blood platelet lumi-aggregometer in 20 newly diagnosed chronic myeloid leukemia patients before and after imatinib mesylate treatment.Results:At the time of diagnosis, 17/20 patients had abnormal platelet aggregation results; 8 (40%) had hypoactivity, 6 (30%) had hyperactivity, and 3 (15%) had mixed hypo- and hyperactivity. Repeat platelet aggregation studies were performed after a mean of 19 months (min: 5 months-max: 35 months) in all patients who received imatinib mesylate during this period. After therapy, 18/20 (90%) patients had abnormal laboratory results; 12 (60%) had hypoactive platelets, 4 (20%) had mixed hypo- and hyperactive platelets, and 2 (10%) had hyperactive platelets. Three of the 8 patients with initial hypoactivity remained hypoactive, while 2 developed a mixed picture, 2 became hyperactive, and 1 normalized. Of the 6 patients with initial hyperactivity, 4 became hypoactive and 2 developed a mixed pattern. All of the 3 patients with initial hypo- and hyperactivity became hypoactive. Finally, 2 of the 3 patients with initial normal platelets became hypoactive while 1 remained normal. There was a significant decrease in ristocetin-induced platelet aggregation after therapy (p<0.001), while platelet aggregation and secretion induced by other agonists showed no difference after treatment (p>0.05).Conclusion:These findings indicate that a significant proportion of chronic myeloid leukemia patients have different patterns of platelet function abnormalities and imatinib mesylate has no effect on these abnormalities, with a significant impairment in ristocetin-induced platelet aggregation.

Suppressive effect of exogenous carbon monoxide on abnormal platelet exocytosis and its molecular mechanism in sepsis

To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism. Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation. (1) Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P < 0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P < 0.05]. (2) Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P < 0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P < 0.05]. (3) Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P < 0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group. LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.

Monitoring Residual Platelet Activity Among Patients With Acute Coronary Syndrome Post-PCI by Modified Impedance Whole Blood Platelet Aggregation and Release Method.

Antiplatelet medicines have been one of the cornerstones in the treatment of patients with acute coronary syndrome (ACS). However, adverse cardiovascular events still occur in some patients on standard antiplatelet therapy. Therefore, a reliable laboratory test to monitor the residual platelet activity (RPA) is urgent. We aim to modify the impedance whole blood platelet aggregation (WBA) assay associated with release assay to monitor RPA, despite antiplatelet therapy and assess their relationship with clinical ischemic events. In this study, RPA was tested in 133 patients with ACS postpercutaneous coronary intervention between 24 and 36 hours after a 300-mg clopidogrel loading dose by modified assay. Then, these patients were followed up for 3 months for clinical ischemic events. Meanwhile, platelet activity of 58 healthy volunteers was also tested by modified assay. Results showed that in modified assay the point of platelet magnified activation time (MAT) and maximal platelet adenosine triphosphate release values (RV) have significant differences between healthy volunteers and patients ([90.86 ± 27.60 seconds] vs [206.44 ± 58.97 seconds] and [2.07 ± 0.64 nmol] vs [0.98 ± 0.49 nmol];P< .001 andP< .001, respectively). During follow-up, 5 patients present ischemic events. Receiver-operator characteristic curve showed that the cutoff values for MAT and RV were 156.5 seconds and 1.05 nmol, respectively, with the sensitivity and specificity of 60.00% and 83.30% and 80.00% and 67.50%, respectively; when MAT combined with RV, the sensitivity can be increased to 100%. Therefore, modified impedance WBA and release assay may be a potentially recommended reliable laboratory assays for monitoring the RPA.

The inhibitory mechanism of crude saponin fraction from Korean Red Ginseng in collagen-induced platelet aggregation

BackgroundKorean Red Ginseng has been used as a traditional oriental medicine to treat illness and to promote health for several thousand years in Eastern Asia. It is widely accepted that ginseng saponins, ginsenosides, are the major active ingredients responsible for Korean Red Ginseng’s therapeutic activity against many kinds of illness. Although the crude saponin fraction (CSF) displayed antiplatelet activity, the molecular mechanism of its action remains to be elucidated. MethodsThe platelet aggregation was induced by collagen, the ligand of integrin αIIβI and glycoprotein VI. The crude saponin’s effects on granule secretion [e.g., calcium ion mobilization and adenosine triphosphate (ATP) release] were determined. The activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAPK, and phosphoinositide 3-kinase (PI3K)/Akt was analyzed by immunoblotting. In addition, the activation of integrin αIIbβIII was examined by fluorocytometry. ResultsCSF strongly inhibited collagen-induced platelet aggregation and ATP release in a concentration-dependent manner. It also markedly suppressed [Ca2+]i mobilization in collagen-stimulated platelets. Immunoblotting assay revealed that CSF significantly suppressed ERK1/2, p38, JNK, PI3K, Akt, and mitogen-activated protein kinase kinase 1/2 phosphorylation. In addition, our fraction strongly inhibited the fibrinogen binding to integrin αIIbβ3. ConclusionOur present data suggest that CSF may have a strong antiplatelet property and it can be considered as a candidate with therapeutic potential for the treatment of cardiovascular disorders involving abnormal platelet function.

Effect of sildenafil on platelet function and platelet cGMP of patients with erectile dysfunction.

To investigate the effect of sildenafil on platelet function and cyclic guanosine monophosphate (cGMP) levels in patients with erectile dysfunction, we evaluated the association between erectile function and platelet responses after administration of 100 mg sildenafil. Erectile responses were monitored after 8 daily doses of the drug. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation and simultaneous adenosine triphosphate (ATP) release and cGMP levels were determined before and after sildenafil therapy. Basal levels for platelet aggregation, ATP release and cGMP were compared with age-matched controls. There was no difference among basal levels of platelet responses between patients and controls, except for ADP-induced platelet aggregation (P = 0.04). It was significantly higher in the patient group. Analysis of the responses to sildenafil revealed that for the patients who showed a positive erectile response, there was a significant increase in platelet cGMP (P = 0.028) and a decrease in ADP-induced platelet aggregation (P = 0.04). However, for those who showed a negative or poor erectile response, there was no change in platelet cGMP levels and platelet functions. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. It is concluded that sildenafil increases platelet cGMP in the patients with positive erectile response. Therefore, it has been speculated that platelet cGMP may be used as an index for erectile response.

Mechanism of platelet activation induced by endocannabinoids in blood and plasma

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB2-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

Levobupivacaine differentially suppresses platelet aggregation by modulating calcium release in a dose-dependent manner

ObjectiveLevobupivacaine, an amide local anesthetic widely used in regional anesthesia, is reported in recent studies that it is a potent inhibitor of platelet functions. However, the concentrations of levobupivacaine were limitedly estimated in these reports. Additionally, the mechanisms by which it affects platelet function and blood coagulation is still not entirely known. The purpose of this study was to further investigate its effects on platelet function and the possible signaling mechanisms under various concentrations of levobupivacaine. MethodsBlood samples collected from healthy volunteers were separated into whole blood, platelet-rich-plasma and washed platelets. The effect of levobupivacaine on platelet aggregation was studied using platelet function analyzer (PFA-100) and platelet aggregometer. Agonist-induced platelet adenosine triphosphate (ATP) release, cytosolic calcium mobilization, thromboxane B2 (TxB2) secretion and platelet P-selectin translocation under various concentrations of levobupivacaine were investigated. ResultsOur results indicated that levobupivacaine possessed negative effect on platelet aggregation. The closure times of (PFA-100) were lengthened and the agonist-induced platelet aggregation was significantly attenuated by levobupivacaine even at a low dose (50 μgml−1). Pretreatment with levobupivacaine produced significant changes in agonist-induced platelet P-selectin translocation, ATP release, thromboxane A2 (TxA2) production, and calcium mobilization in a dose-dependent manner. The p38 mitogen-activated protein kinases (MAPK), protein kinase C (PKC) δ subtype, cytosolic phospholipase A2 (cPLA2), and protein kinase B (PKB or Akt) were involved in collagen-induced platelet signaling, which would be responsible for antiplatelet effects of levobupivacaine. ConclusionWe explored possible targets of levobupivacaine on platelets aggregation signaling mechanisms. Our data revealed that p38 MAPK, PKC δ subtype, cPLA2, and Akt were pathways involved in collagen-induced platelet signaling, which might be responsible for antiplatelet effects of levobupivacaine. Our study did provide direct evidence bolstering the critical mechanisms of levobupivacaine within different contexts. Additionally, levobupivacaine imposed a negative effect on platelet aggregation through multiple signaling pathways.

Acquired cytochrome C oxidase impairment in apheresis platelets during storage: a possible mechanism for depletion of metabolic adenosine triphosphate

Intracellular adenosine triphosphate (ATP) levels decline significantly during storage of platelet (PLT) products, in part due to PLT degranulation. However, metabolic ATP stores also become depleted during storage through an unclear mechanism. Since both anaerobic glycolysis and oxidative phosphorylation are important for PLT ATP production, it is possible that the reduction in metabolic ATP reflects impaired oxidative phosphorylation. To assess this, we evaluated the kinetic activity and protein expression of cytochrome C oxidase (CcOX) in stored apheresis PLTs. Apheresis PLTs were collected and stored with agitation at 22 ± 2°C for 7 days. In vitro measurements of PLT metabolic state, function, and activation were performed on Days 0, 2, 4, and 7 of storage. Total PLT ATP content, steady-state CcOX kinetic activity, and protein immunoblotting for CcOX Subunits I and IV were also performed using isolated PLT mitochondria from simultaneously collected samples. Intra-PLT ATP and steady-state PLT CcOX activity declined significantly and in a progressive manner throughout storage while steady-state levels of CcOX I and IV protein remained unchanged. Time-dependent decline in CcOX activity correlated with progressive ATP depletion over time. During storage of apheresis PLTs for 7 days, the parallel decline in CcOX function and intra-PLT ATP suggests development of an acquired impairment in PLT oxidative phosphorylation associated with perturbed ATP homeostasis in stored PLTs.

Diagnostic Usefulness of a Lumi-Aggregometer Adenosine Triphosphate Release Assay for the Assessment of Platelet Function Disorders

Platelet dense granule release assays are recommended for diagnosing platelet function disorders and are commonly performed by Lumi-Aggregometer (Chrono-Log, Havertown, PA) assays of adenosine triphosphate (ATP) release. We conducted a prospective cohort study of people tested for ATP release defects to assess bleeding symptoms. Reduced release, with 1 or more agonists, was more common among patients with bleeding disorders than among healthy control subjects (P < .001). The respective likelihood (odds ratio [95% confidence interval]) of a bleeding disorder or an inherited platelet function disorder were high when release was reduced with 1 or more agonists (17 [6-46]; 128 [30-545]), even if aggregation was normal (12 [4-34]; 105 [20-565]). ATP release had high specificity and moderate sensitivity for inherited platelet function disorders, with most abnormalities detected by the combination of 6 μmol/L epinephrine, 5.0 μg/mL collagen, and 1 μmol/L U46619. Platelet ATP release assays are useful for evaluating common bleeding disorders, regardless of aggregation findings.

Human Platelet Aggregation and Degranulation Is Induced In Vitro by L-Thyroxine, but Not by 3,5,3′-Triiodo-L-Thyronine or Diiodothyropropionic Acid (DITPA)

The endogenous thyroid hormones L-thyroxine (T(4)) and 3,5,3'-triiodo-L-thyronine (T(3)) induce angiogenesis via an endothelial cell iodothyronine receptor on integrin alphaVbeta3. This receptor also exists on platelets. Diiodothyropropionic acid (DITPA) and GC-1, a noniodinated thyroid hormone analog, also induce angiogenesis. Here we examined the effects of iodothyronines (L-T(4) vs L-T(3)) and analogs DITPA and GC-1 on human platelet function. Subthreshold aggregation of platelets obtained from healthy human donors was induced with collagen. Platelet activation (proaggregation) and adenosine triphosphate (ATP) secretion (degranulation) induced by L-T( 4), L-T(4)-agarose, L-T(3), DITPA, or GC-1 were determined simultaneously. Platelet aggregation and ATP secretion induced by a subthreshold level of collagen were enhanced 3-fold by either L-T(4) or L-T( 4)-agarose (0.01 micromol/L) as compared to control, whereas, L-T( 3), DITPA, or GC-1 had no effect under the same conditions. The platelet proaggregatory and degranulation effects of L-T(4) were blocked by the alphavbeta3 antagonist XT199 (0.1 micromol/ L) and by tetraiodothyroacetic acid (tetrac; 0.1 micromol/L). Tetrac inhibits binding of thyroid hormone analogs to the receptor on alphavbeta3 and lacks thyromimetic activity at this site; thus, the proaggregatory action of L-T(4) likely involves the cell surface receptor on integrin alphavbeta3. The thyroid hormone receptor (TR) on human platelets but not endothelial cells distinguishes among iodothyronines, reflecting quantitative differences in integrin sites on endothelial cells and platelets or qualitative differences in the phospholipids/protein microenvironment of endothelial and platelet membranes that can affect integrin function. Additional studies in different populations with larger sample sizes are warranted to determine the impact of the current findings on clinical interventions.

Effect of Iron Therapy on Platelet Function among Iron-Deficient Women with Unexplained Menorrhagia

This study was performed to evaluate the effect of iron therapy on platelet function among women with unexplained menorrhagia in order to better understand possible interactions between iron deficiency anemia and platelet behavior and menorrhagia. Platelet aggregation and adenosine triphosphate (ATP) release induced by 5.0 mM adenosine diphosphate (ADP), 0.5 mM arachidonic acid (AA), 1.0 mg/ml ristocetin and 2 μg/ml collagen were studied by whole-blood platelet lumi-aggregometer in 50 menorrhagic women before and after oral iron therapy and in 22 women of the control group. There was a significant increase in AA- induced platelet aggregation (p < 0.05) and a decrease in ristocetin-induced platelet aggregation (p < 0.01) after treatment. Pre- and posttreatment platelet aggregation responses to ADP and collagen were not significantly different (p > 0.05). Pre- and posttreatment platelet secretion responses to all agonists disclosed no significant difference (p > 0.05). There was no significant difference between the study group after treatment and the control group in respect to platelet aggregation and ATP secretion values induced by all agonists (p > 0.05). We conclude that iron deficiency anemia in women causes AA-induced platelet dysfunction, which may give rise to increased menstrual blood loss and can be reversed by iron repletion.

Laboratory response to intranasal desmopressin in women with menorrhagia and platelet dysfunction

Intranasal desmopressin (IN-DDAVP) is used for home treatment of menorrhagia in women with inherited bleeding disorders. The effect of IN-DDAVP on laboratory haemostatic parameters in women with menorrhagia related to platelet dysfunction is unknown. We evaluated the effects of IN-DDAVP on haemostatic parameters in women with menorrhagia and platelet dysfunction and correlated them with menstrual flow. Eleven women (aged 18-45) with menorrhagia and haemostatic abnormalities had determination of von Willebrand factor antigen (VWF:Ag), von Willebrand factor ristocetin cofactor (VWF:RCo) activity, factor VIII coagulant activity (FVIII:C), platelet aggregation and platelet adenosine tri-phosphate (ATP) release pre-IN-DDAVP and 60-min post-IN-DDAVP. Eight of eleven women underwent platelet function analyzer (PFA-100) closure time determination with collagen/adrenaline and collagen/adenosine diphosphate cartridges pretreatment and post-treatment. IN-DDAVP was administered during two consecutive menstrual cycles. Menstrual flow was assessed during each cycle using a pictorial blood assessment chart. Treatment with IN-DDAVP resulted in elevated VWF levels and shortened PFA-100 closure time with significant inverse correlation between shortening of PFA-100 closure times and increases in VWF levels. There were also significant inverse correlations between changes in menstrual flow and changes in VWF:Ag (P = 0.02), VWF:RCo (P = 0.04) and FVIII:C (P = 0.006), following treatment. In vitro platelet aggregation and platelet ATP release response did not correct and did not correlate with changes in menstrual flow. Our results demonstrate a correlation between haemostatic parameters and menstrual flow following IN-DDAVP in women with menorrhagia and platelet dysfunction.

Preservation of platelet aggregation and dense granule secretion during extended storage of blood samples in the presence of a synthetic dual inhibitor of factor Xa and thrombin

The clinical significance of platelet function tests may be limited by the use of citrate anticoagulant. We examined the influence of BAPA, a dual inhibitor of factor Xa and thrombin, on platelet responsiveness to agonists when measured between 2 and 48 h after venipuncture. Blood samples from 24 healthy individuals were anticoagulated with citrate or BAPA. Impedance platelet aggregation (IPA) and adenosine triphosphate (ATP) release induced by ADP, collagen and thrombin-receptor activating peptide (TRAP) were determined in whole blood after a storage period between 2 and 48 h after venipuncture. Citrate resulted in significantly reduced collagen or TRAP-induced IPA and ATP release when measured 32 h and 48 h after blood collection. ADP-induced IPA and ATP release in citrated blood dropped significantly between 8 and 24 h. The length of storage of BAPA-anticoagulated blood samples over 48 h had no significant influence on platelet response to collagen and TRAP. In BAPA-anticoagulated blood, ADP-induced IPA and ATP secretion in whole blood were maintained over a storage period of 32 h. No difference in ADP-induced ATP secretion in whole blood anticoagulated with citrate and BAPA was observed, but it was largely suppressed in BAPA-anticoagulated platelet rich plasma. IPA and ATP release remain stable for at least 32 h when whole blood is anticoagulated with a dual inhibitor of factor Xa and thrombin. This would allow shipment of samples for platelet function testing on the following day. Platelet secretion studies in whole blood may include platelet activation by ADP when BAPA is used as anticoagulant.

Estrogen, inflammation, and platelet phenotype

Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain. This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk. To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study. Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine. Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

Laboratory Response to Intranasal Desmopressin in Women with Menorrhagia and Underlying Platelet Dysfunction.

Intranasal desmopressin (IN-DDAVP) is used for home treatment of menorrhagia in women with inherited bleeding disorders. The effect of IN-DDAVP on laboratory hemostatic parameters in women with menorrhagia and associated platelet dysfunction is unknown. We evaluated the effects of IN-DDAVP on hemostatic parameters in women with menorrhagia and platelet dysfunction and correlated them with menstrual blood flow. Eleven women (aged 18–45) with menorrhagia and abnormal platelet aggregation and/or platelet adenosine tri-phosphate (ATP) release had determination of factor VIII coagulant activity (FVIII: C), von Willebrand factor antigen (VWF: Ag), von Willebrand factor ristocetin cofactor (VWF: RCo) activity, platelet aggregation, and platelet ATP release pre and 60 minutes post IN-DDAVP. Eight of eleven women underwent Platelet Function Analyzer (PFA-100) closure time determination with collagen/epinephrine (CEPI) and collagen/adenosine diphosphate (CADP) cartridge pre and post treatment. IN-DDAVP was administered during two consecutive menstrual cycles. Menstrual blood flow was assessed during each cycle using a pictorial blood assessment chart (PBAC). Following administration of IN-DDAVP there was a 2–3 fold increase in FVIII: C, VWF: Ag, and VWF: RCo observed in most patients over baseline levels. Mean FVIII:C increased from 130% ± 34.8% to 218% ± 96.5% (P < 0.007), VWF: Ag increased from 86.9% ± 35.1% to 128.7% ± 64.3% (P < 0.011), and VWF: RCo increased from 89.8% ± 42.8% to 139.6% ± 86.5% (P < 0.02). The median baseline PBAC score of 235 decreased to 136 following IN-DDAVP. In addition, there were significant inverse correlations between changes in PBAC score and changes in VWF: Ag (rs= − 0.85, p = 0.02), VWF: RCo (rs= −0.81, p = 0.029), and FVIII:C (rs= 0.92, p = 0.003) with IN-DDAVP. Mean PFA-100 closure time with the CEPI cartridge shortened from 166.8sec ± 85sec to 90.9sec ± 49.3sec (p< 0.02), and the closure time with the CADP cartridge shortened from 104.5sec ± 34.3sec to 64.8sec ± 40.4sec (p <0.007). There were significant inverse correlations between post IN-DDAVP PFA-100 CADP closure time and post IN-DDAVP FVIII:C (rs= −0.86, p = 0.005), VWF: Ag (rs= −0.72, p = 0.04), and VWF: RCo (rs= −0.80, p = 0.01). In addition there were also significant inverse correlations between post IN-DDAVP PFA-100 CEPI closure time and post IN-DDAVP FVIII:C (rs= −0.73, p = 0.03), and VWF: RCo (rs= −0.74, p = 0.03), but not VWF: Ag (rs= −0.66, p = 0.07). In-vitro platelet aggregation and platelet ATP release response did not correct and did not correlate with changes in menstrual blood flow. Our results demonstrate a correlation between VWF parameters and menstrual blood flow following IN-DDAVP in women with menorrhagia and underlying platelet dysfunction.

Effects of bortezomib on platelet aggregation and ATP release in human platelets, in vitro

Introduction Proteasome inhibitor bortezomib (PS-341) has been the first proteasome inhibitor that has entered clinical trials with its antiproliferative and proapoptotic effects in patients with multiple myeloma. Recent studies indicate that proteasome inhibitors can be useful in prevention of experimental arterial thrombosis in renovascular hypertensive rat models. The aim of the present study is to investigate the effect of bortezomib on in vitro platelet aggregation and adenosine triphosphate (ATP) release of human platelets. Materials and methods For this purpose, platelet aggregation was induced in the platelet-rich plasma (PRP) using 3 μg ml − 1 collagen, 5 μM adenosine diphosphate (ADP), 10 μM epinephrine and 1 U ml − 1 thrombin and ATP release was induced by collagen. Results and conclusions Bortezomib showed an inhibitory effect on platelet aggregation induced by ADP in human PRP in a dose- and time-dependent manner, whereas it had no effect on collagen-, epinephrin and thrombin-induced aggregation. ATP-release reaction induced by collagen was inhibited dose- and time-dependently by bortezomib, even though collagen-induced platelet aggregation was apparently not affected in human PRP. These findings indicate that bortezomib may be an antiaggregating agent and its' effects may be related to adenine nucleotide receptor dependent regulatory proteins which are important for physiological and pathophysiological cellular processes. However, our in vitro studies suggest that this hypothesis is inadequate to explain the observations completely. This phenomenon and its clinical implication justify further clinical investigations.