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1.  The Relationship between Fractional Flow Reserve, Platelet Reactivity and Platelet Leukocyte Complexes in Stable Coronary Artery Disease 
PLoS ONE  2013;8(12):e83198.
Background
The presence of stenoses that significantly impair blood flow and cause myocardial ischemia negatively affects prognosis of patients with stable coronary artery disease. Altered platelet reactivity has been associated with impaired prognosis of stable coronary artery disease. Platelets are activated and form complexes with leukocytes in response to microshear gradients caused by friction forces on the arterial wall or flow separation. We hypothesized that the presence of significantly flow-limiting stenoses is associated with altered platelet reactivity and formation of platelet-leukocyte complexes.
Methods
One hundred patients with stable angina were studied. Hemodynamic significance of all coronary stenoses was assessed with Fractional Flow Reserve (FFR). Patients were classified FFR-positive (at least one lesion with FFR≤0.75) or FFR-negative (all lesions FFR>0.80). Whole blood samples were stimulated with increasing concentrations of ADP, TRAP, CRP and Iloprost with substimulatory ADP. Expression of P-selectin as platelet activation marker and platelet–leukocyte complexes were measured by flowcytometry. Patients were stratified on clopidogrel use. FFR positive and negative patient groups were compared on platelet reactivity and platelet-leukocyte complexes.
Results
Platelet reactivity between FFR-positive patients and FFR-negative patients did not differ. A significantly lower percentage of circulating platelet-neutrophil complexes in FFR-positive patients and a similar non-significant decrease in percentage of circulating platelet-monocyte complexes in FFR-positive patients was observed.
Conclusion
The presence of hemodynamically significant coronary stenoses does not alter platelet reactivity but is associated with reduced platelet-neutrophil complexes in peripheral blood of patients with stable coronary artery disease.
doi:10.1371/journal.pone.0083198
PMCID: PMC3877034  PMID: 24391745
2.  The effect of clopidogrel on platelet activity in patients with and without type-2 diabetes mellitus: a comparative study 
Background
Although antiplatelet therapy involving clopidogrel is a standard treatment for preventing cardiovascular events after coronary stent implantation, patients can display differential responses. Here, we assessed the effectiveness of clopidogrel on platelet function inhibition in subjects with and without type-2 diabetes and stable coronary artery disease. In addition, we investigated the correlation between platelet function and routine clinical parameters.
Methods
A total of 64 patients with stable coronary heart disease were enrolled in the study. Among these, 32 had known type-2 diabetes, whereas the remaining 32 subjects were non-diabetics (control group). A loading dose of 300 mg clopidogrel was given to clopidogrel-naïve patients (13 patients in the diabetes group and 14 control patients). All patients were given a daily maintenance dose of 75 mg clopidogrel. In addition, all patients received 100 mg ASA per day. Agonist-induced platelet aggregation measurements were performed on hirudin-anticoagulated blood using an impedance aggregometer (Multiple Platelet Function Analyzer, Dynabyte, Munich, Germany). Blood samples were drawn from the antecubital vein 24 h after coronary angiography with percutaneous coronary intervention. The platelets were then stimulated with ADP alone or ADP and prostaglandin-E (ADP and ADP-PGE tests, respectively) in order to evaluate clopidogrel-mediated inhibition of platelet function. The effectiveness of ASA was measured by stimulation with arachidonic acid (ASPI test). In addition, maximal platelet aggregation was assessed via stimulation with thrombin receptor-activating peptide (TRAP test).
Results
Patients with diabetes exhibited significantly less inhibition of platelet function than patients without diabetes (ADP-PGE test p = 0.003; ASPI test p = 0.022). Administering a clopidogrel loading dose of 300 mg did not result in a lower level of ADP-PGE-induced platelet reactivity in comparison to the use of a 75 mg maintenance dose. Moreover, we observed that ADP-PGE-induced platelet inhibition was positively correlated with fasting blood glucose and HbA1c (p < 0.01).
Conclusions
Patients with type-2 diabetes exhibited increased platelet reactivity compared to patients without diabetes despite combined treatment with clopidogrel and ASA. Using a loading dose of clopidogrel rather than small daily doses was not sufficient for adequately overcoming increased platelet reactivity in patients with type-2 diabetes, highlighting the need for more effective anti-platelet drugs for such patients.
doi:10.1186/s12933-015-0182-7
PMCID: PMC4324649  PMID: 25645908
Clopidogrel; Diabetes; Platelet function; Ccoronary heart disease; Percutaneous coronary intervention
3.  Differences in Platelet Function In Patients with Acute Myeloid Leukaemia and Myelodysplasia Compared to Equally Thrombocytopenic Patients with Immune Thrombocytopenia 
Background
Severe thrombocytopenia is a major risk factor for haemorrhage, and yet platelet function and bleeding risk at low platelet counts are poorly understood because of limitations of platelet function testing at very low platelet counts.
Objectives
To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS) to patients with immune thrombocytopenia (ITP).
Methods
Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms.
Results
Compared with patients with ITP, patients with AML/MDS had smaller platelets, lower IPF, and substantially lower platelet surface expression of activated GPIIb/IIIa and GPIb both with and without addition of ex vivo ADP or TRAP. In both ITP and AML/MDS, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb/IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than those with no bleeding.
Conclusions
AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly-produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia.
doi:10.1111/j.1538-7836.2011.04506.x
PMCID: PMC3210015  PMID: 21920014
Autoimmunity; bleeding; flow cytometry; haemorrhage; thrombocytopenia; thrombopoiesis
4.  Increased platelet reactivity in patients with late-stage metastatic cancer 
Cancer Medicine  2013;2(4):564-570.
Abstract
Platelet hyperreactivity is associated with an increased risk of thrombosis. Cancer patients are at an increased risk of thrombosis, a risk that increases with disease progression. While cancer patients show evidence of platelet activation in vivo, few studies have extensively assessed whether these patients display platelet hyperreactivity. We hypothesized that patients with metastatic cancer would display platelet hyperreactivity, reflecting their associated high risk of thrombosis. In a cohort of patients with metastatic cancer (n = 13), we assessed platelet function using well-established assays of platelet reactivity (agonist-induced platelet aggregation, spontaneous platelet aggregation, and agonist-induced P-selectin expression). In comparison with healthy controls (n = 10), patients with metastatic cancer displayed global platelet hyperreactivity. Agonist-induced platelet aggregation responses to ADP (adenosine diphosphate), epinephrine, collagen, arachidonic acid, and PAR-1 (protease-activated receptor-1) activating peptide, as well as spontaneous platelet aggregation, were significantly increased in patients with metastatic cancer. Furthermore, agonist-induced platelet P-selectin expression was also significantly increased within the patient cohort. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis.
We assessed platelet function in a cohort of patients with metastatic cancer (n = 13) using well-established assays of platelet reactivity. Agonist-induced platelet aggregation and activation in response to platelet agonists, as well as spontaneous platelet aggregation, was significantly increased in cancer patients compared with healthy controls. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis.
doi:10.1002/cam4.86
PMCID: PMC3799291  PMID: 24156029
Activation; aggregation; hyperreactivity; metastasis; platelets
5.  Increased Platelet Reactivity in Idiopathic Pulmonary Fibrosis Is Mediated by a Plasma Factor 
PLoS ONE  2014;9(10):e111347.
Introduction
Idiopathic Pulmonary Fibrosis (IPF) is a progressive, incurable fibrotic interstitial lung disease with a prognosis worse than many cancers. Its pathogenesis is poorly understood. Activated platelets can release pro-fibrotic mediators that have the potential to contribute to lung fibrosis. We determine platelet reactivity in subjects with IPF compared to age-matched controls.
Methods
Whole blood flow cytometry was used to measure platelet-monocyte aggregate formation, platelet P-selectin expression and platelet fibrinogen binding at basal levels and following stimulation with platelet agonists. A plasma swap approach was used to assess the effect of IPF plasma on control platelets.
Results
Subjects with IPF showed greater platelet reactivity than controls. Platelet P-selectin expression was significantly greater in IPF patients than controls following stimulation with 0.1 µM ADP (1.9% positive ±0.5 (mean ± SEM) versus 0.7%±0.1; p = 0.03), 1 µM ADP (9.8%±1.3 versus 3.3%±0.8; p<0.01) and 10 µM ADP (41.3%±4.2 versus 22.5%±2.6; p<0.01). Platelet fibrinogen binding was also increased, and platelet activation resulted in increased platelet-monocyte aggregate formation in IPF patients. Re-suspension of control platelets in plasma taken from subjects with IPF resulted in increased platelet activation compared to control plasma.
Conclusions
IPF patients exhibit increased platelet reactivity compared with controls. This hyperactivity may result from the plasma environment since control platelets exhibit increased activation when exposed to IPF plasma.
doi:10.1371/journal.pone.0111347
PMCID: PMC4206466  PMID: 25338090
6.  In vitro norepinephrine significantly activates isolated platelets from healthy volunteers and critically ill patients following severe traumatic brain injury 
Critical Care  2008;12(3):R80.
Introduction
Norepinephrine, regularly used to increase systemic arterial blood pressure and thus improve cerebral perfusion following severe traumatic brain injury (TBI), may activate platelets. This, in turn, could promote microthrombosis formation and induce additional brain damage.
Methods
The objective of this study was to investigate the influence of norepinephrine on platelets isolated from healthy volunteers and TBI patients during the first two post-traumatic weeks. A total of 18 female and 18 male healthy volunteers of different age groups were recruited, while 11 critically ill TBI patients admitted consecutively to our intensive care unit were studied. Arterial and jugular venous platelets were isolated from norepinephrine-receiving TBI patients; peripheral venous platelets were studied in healthy volunteers. Concentration-dependent functional alterations of isolated platelets were analyzed by flow cytometry, assessing changes in surface P-selectin expression and platelet-derived microparticles before and after in vitro stimulation with norepinephrine ranging from 10 nM to 100 μM. The thrombin receptor-activating peptide (TRAP) served as a positive control.
Results
During the first week following TBI, norepinephrine-mediated stimulation of isolated platelets was significantly reduced compared with volunteers (control). In the second week, the number of P-selectin- and microparticle-positive platelets was significantly decreased by 60% compared with the first week and compared with volunteers. This, however, was associated with a significantly increased susceptibility to norepinephrine-mediated stimulation, exceeding changes observed in volunteers and TBI patients during the first week. This pronounced norepinephrine-induced responsiveness coincided with increased arterio-jugular venous difference in platelets, reflecting intracerebral adherence and signs of cerebral deterioration reflected by elevated intracranial pressure and reduced jugular venous oxygen saturation.
Conclusion
Clinically infused norepinephrine might influence platelets, possibly promoting microthrombosis formation. In vitro stimulation revealed a concentration- and time-dependent differential level of norepinephrine-mediated platelet activation, possibly reflecting changes in receptor expression and function. Whether norepinephrine should be avoided in the second post-traumatic week and whether norepinephrine-stimulated platelets might induce additional brain damage warrant further investigations.
doi:10.1186/cc6931
PMCID: PMC2481479  PMID: 18564410
7.  Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study 
Background
Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.
Methods
With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.
Results
The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone.
Conclusion
The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.
doi:10.1186/1479-5876-7-42
PMCID: PMC2699331  PMID: 19508722
8.  Plasma and Whole Blood Clot Strength Measured by Thrombelastography in Patients Treated with Clopidogrel during Acute Coronary Syndromes 
Thrombosis research  2013;132(2):e94-e98.
Introduction
Treatment with clopidogrel, a selective platelet P2Y12 receptor antagonist, reduces risk of recurrent ischemic events in patients with acute coronary syndrome (ACS), by limiting platelet aggregation and activation. Stable whole blood clot formation requires activation of platelets, generation of fibrin and final fibrin crosslinks. In this study we intended to compare plasma and whole blood thrombelastography (TEG) measurements in patients during ACS.
Materials and Methods
Whole blood and plasma samples from 32 patients with nonST segment elevation myocardial infarction (NSTEMI) were collected after administration of clopidogrel. Whole blood and plasma fibrin clot strength (MA) were determined by TEG. Platelet aggregation was determined by light transmittance aggregometry (LTA) using adenosine 5′-diphosphate (ADP), thrombin receptor activation peptide (TRAP), or collagen as agonists. Fibrinogen and C-reactive protein (CRP) concentrations were measured by ELISA.
Results
Heightened plasma fibrin clot strength was associated with increased platelet reactivity stimulated by ADP (ρ=0.536; p=0.002), TRAP (ρ=0.481; p=0.007), and collagen (ρ=0.538; p=0.01). In contrast to plasma fibrin MA, whole blood MA did not correlate with platelet aggregation. Platelet count was the primary contributor to the difference in thrombin induced whole blood MA and plasma fibrin MA. Increasing levels of CRP were associated with increased plasma fibrin clot strength and platelet reactivity.
Conclusions
Our data suggest that inflammation is associated with increased plasma fibrin clot strength and lower platelet inhibition by clopidogrel during ACS. Platelet count is main contributor to additional contractile force of whole blood TEG as compared to plasma TEG during treatment with clopidogrel.
doi:10.1016/j.thromres.2013.07.012
PMCID: PMC4028119  PMID: 23920429
CRP; platelet; clopidogrel; thrombelastography; coagulation
9.  Platelet Activity and Sensitivity to Agonists After Exhaustive Treadmill Exercise 
The extent of platelet activation after exhaustive exercise remains under discussion. Previous studies have provided contrary data, probably due to differences in the methodologies and the enrolled subjects. In the present study a maximal treadmill exercise (TR) was used to test platelet activity and -reactivity in 13 healthy non-smoking men. Blood samples were taken after a 30min rest, immediately before and after exercise, and 1h after completion of exercise. Platelets were analysed by whole blood flow cytometry either directly or after in vitro stimulation by incubating the blood samples for 10min with TRAP-6 (10µM) or ADP (5µM or 2,5µM). Binding of an anti-CD62P antibody or a PAC1 antibody directed against the activated fibrinogen receptor GPIIb/IIIa were used as a measure of platelet activation. Immediately after TR the percent CD62P positive platelets (%PC) unstimulated increased (p<0.01) from 0.77±0.06 to 1.12± 0.09 %PC and in PAC1 (p<0.05) from 2.32 ±0.54 to 3.83±0.81 %PC (mean±SEM). After ADP-stimulation an increase from 4.18±1.02 to 5.69±1.40 %PC in CD62P (p<0.01) and from 45.7±3.4 to 57.9±6.6 %PC in PAC1 (p<0.05) after TR were detected. Using TRAP-6-stimulation only the increase of PAC1 (p<0.01) after TR was different in comparison with the control experiment without exercise. Soluble CD62P in plasma as a marker of platelet and endothelial cell activation was also enhanced (p<0.05) after TR. Although these results indicate that exhaustive exercise lead to a small platelet activation and an increase in platelet reactivity, it is rather doubtful that these changes alone implicate a prothrombotic situation in healthy young non-smokers.
PMCID: PMC3937570  PMID: 24616605
Platelet activation; CD62P; PAC1; sCD62P; physical activity
10.  Nitric oxide released from activated platelets inhibits platelet recruitment. 
Journal of Clinical Investigation  1997;100(2):350-356.
Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.
PMCID: PMC508197  PMID: 9218511
11.  Lack of influence of the COX inhibitors metamizol and diclofenac on platelet GPIIb/IIIa and P-selectin expression in vitro 
BMC Anesthesiology  2004;4:4.
Background
The effect of non-steroidal anti-inflammatory drugs (NSAIDs) for reduced platelet aggregation and thromboxane A2 synthesis has been well documented. However, the influence on platelet function is not fully explained. Aim of this study was to examine the influence of the COX-1 inhibiting NSAIDs, diclofenac and metamizol on platelet activation and leukocyte-platelet complexes, in vitro. Surface expression of GPIIb/IIIa and P-selectin on platelets, and the percentage of platelet-leukocyte complexes were investigated.
Methods
Whole blood was incubated with three different concentrations of diclofenac and metamizol for 5 and 30 minutes, followed by activation with TRAP-6 and ADP. Rates of GPIIb/IIIa and P-selectin expression, and the percentage of platelet-leukocyte complexes were analyzed by a flow-cytometric assay.
Results
There were no significant differences in the expression of GPIIb/IIIa and P-selectin, and in the formation of platelet-leukocyte complexes after activation with ADP and TRAP-6, regarding both the time of incubation and the concentrations of diclofenac and metamizol.
Conclusions
Accordingly, the inhibitory effect of diclofenac and metamizol on platelet aggregation is not related to a reduced surface expression of P-selectin and GPIIb/IIIa on platelets.
doi:10.1186/1471-2253-4-4
PMCID: PMC411036  PMID: 15107131
diclofenac; metamizol; platelet; P-selectin; GPIIb/IIIa; flow cytometry
12.  Whole Blood Platelet Aggregation and Release Reaction Testing in Uremic Patients 
BioMed Research International  2013;2013:486290.
Background. Platelet function analysis utilizing platelet-rich plasma and optical density based aggregometry fails to identify patients at risk for uremia associated complications. Methods. We employed whole blood platelet aggregation analysis based on impedance as well as determination of ATP release from platelet granules detected by a chemiluminescence method. Ten chronic kidney disease (CKD) stage 4 or 5 predialysis patients underwent platelet evaluation. Our study aims to evaluate this platform in this patient population to determine if abnormalities could be detected. Results. Analysis revealed normal aggregation and ATP release to collagen, ADP, and high-dose ristocetin. ATP release had a low response to arachidonic acid (0.37 ± 0.26 nmoles, reference range: 0.6–1.4 nmoles). Platelet aggregation to low-dose ristocetin revealed an exaggerated response (20.9 ± 18.7 ohms, reference range: 0–5 ohms). Conclusions. Whole blood platelet analysis detected platelet dysfunction which may be associated with bleeding and thrombotic risks in uremia. Diminished ATP release to arachidonic acid (an aspirin-like defect) in uremic patients may result in platelet associated bleeding. An increased aggregation response to low-dose ristocetin (a type IIb von Willebrand disease-like defect) is associated with thrombus formation. This platelet hyperreactivity may be associated with a thrombotic diathesis as seen in some uremic patients.
doi:10.1155/2013/486290
PMCID: PMC3708384  PMID: 23878808
13.  Differences in responses of platelets to fluid shear stress in patients with peripheral artery disease (PAD) and coronary artery disease (CAD) 
Platelets  2009;20(3):199-205.
Information on differences in platelet function between patients with peripheral arterial disease (PAD) and patients with coronary artery disease (CAD) is limited. We sought to examine the differences in the platelets response to shear stress in patients with PAD compared to those with CAD. Men with symptomatic PAD (ankle brachial index [ABI] <0.9; n=29) were compared with similarly aged men with CAD (post coronary artery bypass grafting; n=40) but without PAD. All participants were on aspirin, and none were on clopidogrel. We measured changes in shear-induced platelet aggregation (SIPA) and shear-induced P-selectin expression (SIPE) under fluid shear rates of 5,000 and 10,000 sec-1 which are typically found in arterioles and stenosed arteries, respectively. Aggregation was also induced by a combined stimulation of collagen, fluid shear stress, and adenosine diphosphate (ADP) or epinephrine using a platelet function analyzer (PFA-100) as well as optical aggregometry (arachidonic acid, collagen and epinephrine). Analyses of covariance adjusted for age, aspirin dose, and statin use were used to estimate differences between the groups. Values of SIPA at fluid shear rates of 5,000 and 10,000 sec-1 were significantly higher in the PAD group, while there were no differences between the PAD and CAD groups in SIPE at both fluid shear rates. However, baseline shear-induced P-selectin expression was higher in patients with PAD than CAD (mean fluorescence intensity [MFI]=2.93 ±1.37 vs.1.94 ±0.67; p=0.01), while the percentage increases in SIPA and SIPE at fluid shear rates of 5,000 and 10,000 sec-1 were significantly higher in patients with CAD when compared to PAD (p <0.001 for all comparisons). Although there were several similarities in platelet function between men with PAD and men with CAD, significant differences in platelet responses to shear stress were observed in men with PAD when compared to those with CAD. Although the mechanism for these observed differences are not clear, we hypothesize that in vivo platelet activation in PAD patients may contribute to the differences and will need to be further investigated.
doi:10.1080/09537100902780643
PMCID: PMC2869211  PMID: 19437338
PAD; CAD; Platelet function; shear
14.  Amphotericin B induced abnormalities in human platelets 
Clinical Molecular Pathology  1996;49(5):M301-M307.
Aims—To investigate in vitro the effect of amphotericin B on platelets in order to understand poor platelet recovery in patients receiving platelet transfusions and amphotericin B simultaneously.
Methods—Washed platelets were isolated from platelet concentrates and exposed to amphotericin B (4 μg/ml) for one hour. Platelet function was assessed by aggregation response to thrombin (0-0.6 U/ml), serotonin release, response to hypotonic stress, and mean platelet volume. The expression of surface membrane glycoprotein (GP) Ib-IX complex, GPIIb-IIIa complex and CD62P (P-selectin) was examined by flow cytometry using fluorescence labelled monoclonal antibodies. Heterotypic cell adhesion was measured in amphotericin B treated platelets coincubated with isolated, autologous polymorphonuclear leucocytes (PMN) by flow cytometric analysis.
Results—Amphotericin B induced platelet dysfunction. The rate of aggregation by thrombin, serotonin uptake and thrombin induced release of serotonin, and the response of platelets to hypotonic stress were inhibited. There was up to a two-fold increase in the mean platelet volume. The expression of platelet surface GPIb-IX and GPIIb-IIIa was not affected. P-selectin, normally expressed only on the surface of activated platelets, was also expressed on unactivated platelets. Amphotericin B increased platelet adherence to PMN and the number of platelets bound per PMN.
Conclusions—In vitro, amphotericin B induces P-selectin expression on the surface of unactivated platelets and increases platelet adhesion to PMN, which is exacerbated by storage. Platelet dysfunction resulting from exposure to amphotericin B may contribute to poor platelet recovery in vivo when amphotericin B is administered concomitantly with platelet transfusion.
PMCID: PMC408077  PMID: 16696093
amphotericin B; platelets; surface membrane glycoprotein; flow cytometry
15.  A Flow Cytometric Analysis of the Inhibition of Platelet Reactivity Due to Nitrite Reduction by Deoxygenated Erythrocytes 
PLoS ONE  2014;9(3):e92435.
Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects.
doi:10.1371/journal.pone.0092435
PMCID: PMC3958531  PMID: 24642865
16.  Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients 
Summary
Background
Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients.
Methods
We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient.
Results
Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage.
Discussion
Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients.
doi:10.1159/000350688
PMCID: PMC3638932  PMID: 23652405
Flow cytometry; Platelet, Platelet activation; Platelet concentrates; Platelet function; Platelet storage; Platelet transfusion; Platelets; Thrombocytopenia
17.  The influence of Erythropoietin on platelet activation, thrombin generation and FVII/active FVII in patients with AMI 
Thrombosis Journal  2014;12:18.
Background
Erythropoietin (Epo) has been shown to improve myocardial function in models of experimental myocardial infarction, but has also been associated with a rise in thromboembolic events. Thus, the aim of this study was to investigate the influence of Epo on platelet activation and coagulation in patients with acute myocardial infarction (AMI).
Methods
The study was designed as a substudy of the randomised, double-blind, placebo controlled REVIVAL-3 (REgeneration of VItal Myocardium in ST-Segment EleVation MyocardiAL Infarction by Erythropoietin) study that investigated the effects of recombinant human Epo in AMI. Serial venous blood samples were collected before and after study medication. Circulating prothrombin fragment F1 + 2, FVII, active FVII, beta thromboglobulin (TG) and P-Selectin were measured before and 60 hours after randomization by immunoassay (n = 94). In a randomly selected subgroup platelet aggregation was measured using whole blood aggregometry (Multiplate Analyzer, n = 45).
Results
After 5 days an increase in FVII was observed after Epo as compared to placebo (P = 0.02), yet active FVII and prothrombin fragment F1 + 2 remained unchanged. Moreover, no statistically significant differences in circulating TG or P-selectin were observed between the groups. As an expected response to peri-interventional therapy with clopidogrel and aspirin, platelet aggregation after stimulation with ADP, TRAP, ASPI or collagen decreased 12 hours and 2 days after PCI. However, no difference between the Epo and the placebo group was observed.
Conclusion
After treatment with Epo in patients with AMI a slight increase in circulating FVII after Epo was not associated with an increase in active FVII, prothrombin fragment F1 + 2, TG or P-selectin. Moreover, platelet aggregation was not altered after treatment with Epo as compared to placebo.
Trial registration
ClinicalTrials.gov Identifier: NCT01761435
doi:10.1186/1477-9560-12-18
PMCID: PMC4165375  PMID: 25228850
Platelet activation; Erythropoietin; AMI; PCI
18.  Platelet Counts and Platelet Activation Markers in Obese Subjects 
Mediators of Inflammation  2008;2008:834153.
Objective. In this work we studied the correlation between platelet count, platelet activation, and systemic inflammation in overweight, obese, and morbidly obese individuals. Methods and subjects. A total of 6319 individuals participated in the study. Complete blood counts, high sensitivity C-reactive protein (hs-CRP) serum levels, and body mass index (BMI) were measured during routine checkups. Platelet activation markers were studied among 30 obese (BMI = 41 ± 8 kg/m2) and 35 nonobese (BMI = 24 ± 3 kg/m2) individuals. Platelet activation status was evaluated by flow cytometry using specific antibodies against the activated platelet membrane glycoprotein IIb/IIIa, p-selectin (CD-62 p), and binding of Annexin-V to platelet anionic phospholipids. Results. Overweight, obese, and morbidly obese females had significantly elevated platelet counts ( P < .0001) compared with normal-weight females. No significant elevation of platelet counts was observed in the male subgroups. A significant age adjusted correlation between BMI and platelet counts ( P < .0001) was found among females. This correlation was attenuated (P = .001) after adjustment for hs-CRP concentrations. The flow cytometry analysis of platelets showed no significant differences in activation marker expression between nonobese and obese individuals. Discussion. Obesity may be associated with elevated platelet counts in females with chronic inflammation. Obesity is not associated with increased platelet activation.
doi:10.1155/2008/834153
PMCID: PMC2276682  PMID: 18385810
19.  Prednisolone exerts exquisite inhibitory properties on platelet functions 
Biochemical Pharmacology  2012;83(10):1364-1373.
Graphical abstract
We have previously reported presence of the glucocorticoid (GC) receptor (GR) alpha on blood platelets, and its ability to modulate platelet aggregation when activated by the synthetic GC prednisolone (Pred). In the present study we investigated the effects of Pred on broader aspects of platelet functions to unveil novel non-genomic actions on this cell type. Using whole blood assay we demonstrated that Pred was the only GC able to inhibit platelet aggregation and platelet–monocyte interactions. This latter effect was due to regulation of platelets, not monocytes. We next examined the effects of Pred on physiological actions of platelets, observing inhibition of platelet adhesion and spreading on collagen under static conditions. Moreover Pred inhibited thrombus formation under flow, suggesting potential important effects in haemostasis and thrombosis. Pred was unable to regulate platelet reactivity under conditions where the effects of platelet-derived ADP and TxA2 were blocked, suggesting that the GC targeted the activation-dependent component of the adhesion and aggregation response. The effects of Pred were not mediated through cyclic nucleotide signaling, but rather seemed to evolve around selective regulation of P2Y12 ADP receptor signaling, intimating a novel mode of action. This study details the actions of Pred on platelets unveiling novel properties which could be relevant for this GC in controlling unwanted vascular and thrombotic diseases.
doi:10.1016/j.bcp.2012.02.006
PMCID: PMC3320711  PMID: 22366284
Platelet biology; Prednisolone; Glucocorticoid receptor; Adhesion; Aggregation; Cell-to-cell interaction
20.  No Effect of Clopidogrel Activity or Cessation on Vascular Function or Markers of Inflammation 
The platelet adenosine diphosphate (ADP)-receptor blocker clopidogrel is effective in reducing the rate of thrombosis in cardiovascular disease, but may also have nonplatelet activity. However, there is variability in the suppression of platelet function in individuals, leading to the concept of clopidogrel resistance, that is, reduced platelet-suppressing activity. We tested the hypothesis that some of the beneficial effect of clopidogrel may be due to the variable activity of this drug on the vascular system (assessed by plasma markers von Willebrand factor and soluble E-selectin, and functional arterial pulse wave velocity) and inflammation (C-reactive protein and interleukin-6) while 32 patients with coronary artery disease taking 75 mg clopidogrel daily, and again 2 weeks after cessation of clopidogrel therapy. Platelet responsiveness to clopidogrel was assessed by the phosphorylation of intracellular regulatory protein—vasodilator-stimulated phosphoprotein method and aggregometry to ADP. Response to aspirin was assessed using arachidonic acid (AA), and soluble P-selectin and PAC-1 were also measured. While on clopidogrel, there were no relationships between any vascular or inflammatory index and the response to clopidogrel. After stopping clopidogrel, there were no differences in platelet aggregation to AA, or the expression of P-selectin or PAC-1 at rest, or after stimulation by AA, but platelet responses to ADP all increased (p < 0.01). Although soluble P-selectin increased when clopidogrel was stopped (p = 0.006), there were no changes in plasma markers or vascular function. We conclude that 75 mg/day clopidogrel has no effect of markers of vascular function or inflammation.
doi:10.1055/s-0032-1328777
PMCID: PMC3578614  PMID: 24293976
clopidogrel; platelet aggregation; vascular function; inflammation; coronary artery disease; platelet aggregation
21.  Leukocyte Count is Associated with Increased Platelet Reactivity and Diminished Response to Aspirin in Healthy Individuals with a Family History of Coronary Artery Disease 
Thrombosis research  2009;124(3):311-317.
Background
Markers of systemic inflammation, including blood leukocyte count, are associated with increased cardiovascular risk, but the mechanisms underlying this association are unclear. Leukocytes may promote platelet reactivity and thrombus formation, providing a basis for increased risk, but a relation between leukocyte count and platelet function has not been studied.
Methods
We evaluated the relation of blood leukocyte count, C-reactive protein (CRP), and interleukin-6 (IL-6) to platelet aggregation to collagen, ADP and arachidonic acid, and to urinary excretion of 11-dehydro thromboxane B2. Studies were conducted in 1600 individuals (45.0 ± 12.9 years, 42.7% male) at risk for coronary artery disease (CAD) before and after low dose aspirin.
Results
At baseline, platelet reactivity increased with increasing quartile of leukocyte count (median counts for each quartile were normal) for all measures of platelet function (P<0.0001). These relations were unchanged by aspirin. The relation between leukocyte count and each measure of platelet reactivity remained significant (P<0.05) after multivariable adjustment for CRP, IL-6, cardiac risk factors, hematologic variables, and platelet thromboxane production. CRP and IL-6 were independently associated with few measures of platelet reactivity.
Conclusions
Increasing quartile of leukocyte count, even within the normal range, is associated with increasing platelet reactivity in individuals at risk for CAD. This relationship is not altered by aspirin and is independent of inflammatory markers and platelet thromboxane production. Additional studies are needed to determine the mechanism(s) for this association and therapies to reduce cardiovascular risk in patients with elevated leukocyte counts.
doi:10.1016/j.thromres.2008.12.031
PMCID: PMC2763116  PMID: 19185906
coronary disease; leukocytes; myocardial infarction; platelets; thrombosis
22.  Reduced Platelet Thromboxane Formation in Uremia. EVIDENCE FOR A FUNCTIONAL CYCLOOXYGENASE DEFECT 
Journal of Clinical Investigation  1983;71(3):762-768.
A qualitative platelet abnormality and a bleeding tendency are frequently associated with renal failure and uremia. We demonstrated previously that uremic patients display an abnormal platelet aggregation to arachidonic acid and reduced malondialdehyde production in response to thrombin and arachidonic acid. The objectives of this investigation were: (a) to compare platelet prostaglandin (PG) and thromboxane (TX) production in whole blood and in platelet-rich plasma (PRP) of 21 uremic patients and 22 healthy subjects; (b) to evaluate the concentration and activity of platelet PG- and TX-forming enzymes; (c) to assess the functional responsiveness of the platelet TXA2/PGH2 receptor; (d) to explore the hemostatic consequences of partially reduced TXA2 production.
Platelet immunoreactive TXB2 production during whole blood clotting was significantly reduced, by ∼60%, in uremic patients as compared to age- and sex-matched controls. Exogenous thrombin (5-30 IU/ml) failed to restore normal TXB2 production in uremic platelets. Uremic PRP produced comparable or slightly higher amounts of TXB2 than normal PRP at arachidonate concentrations 0.25-1 mM. However, when exposed to substrate concentrations >2 mM, uremic PRP produced significantly less TXB2 than normal PRP. To discriminate between reduced arachidonic acid oxygenation and altered endoperoxide metabolism, the time course of immunoreactive TXB2 and PGE2 production was measured during whole blood clotting. The synthesis and release of both cyclooxygenase-derived products was slower and significantly reduced, at all time intervals considered. Furthermore, PGI2 production in whole blood, as reflected by serum immunoreactive 6-keto-PGF1α concentrations, was significantly reduced in uremic patients as compared with healthy subjects. PGH synthase levels, as determined by an immunoradiometric assay, were not significantly different in platelets from uremic patients as compared to control platelets. A single 40-mg dose of aspirin given to five healthy volunteers reduced their serum TXB2 to levels found in uremic patients. This was associated with a significant increase of threshold aggregating concentrations of ADP and arachidonic acid and prolongation of bleeding time. Substantially similar threshold concentrations of U46619, a TXA2 agonist, induced aggregation of normal and uremic platelets. Prostacyclin induced a significant elevation of uremic platelet cyclic AMP, which was suppressed by U46619, further suggesting normal responsiveness of the TXA2/PGH2 receptor.
We conclude that: (a) an abnormality of platelet arachidonic acid metabolism exists in uremia, leading to a reduced TXA2 production; (b) the characteristics of this abnormality are consistent with a functional cyclooxygenase defect; (c) reduced TXA2 production may partially explain the previously described abnormality of platelet function in uremia.
PMCID: PMC436927  PMID: 6298281
23.  Exposure of platelet fibrinogen receptors by ADP and epinephrine. 
Journal of Clinical Investigation  1979;64(5):1393-1401.
The role of fibrinogen as a cofactor for platelet aggregation was examined by measuring the binding of 125I-labeled human fibrinogen to gel-filtered human platelets both before and after platelet stimulation by ADP and epinephrine. Platelet stimulation by ADP resulted in the rapid, reversible binding of fibrinogen to receptors on the platelet surface. Fibrinogen binding increased as the concentration of ADP was increased from 0.1 to 2 microM, reaching a plateau at higher ADP concentrations. Binding occurred only after platelet stimulation and in the presence of divalent cations. However, fibrinogen binding did not occur to ADP-stimulated platelets from three patients with Glanzmann's thrombasthenia. Analysis of fibrinogen binding as a function of increasing fibrinogen concentration demonstrated that maximal platelet stimulation exposed approximately or equal to 45,000 binding sites per platelet with a dissociation constant of 80--170 nM. These fibrinogen binding parameters were essentially the same whether ADP or epinephrine was the platelet-stimulating agent. Thus, these studies demonstrate that platelet stimulation by ADP and epinephrine exposes a limited number of fibrinogen receptors on the platelet surface. Furthermore, these data suggest that the fibrinogen molecules bound to the platelet as a consequence of platelet stimulation are directly involved in the platelet aggregation response.
Images
PMCID: PMC371288  PMID: 574143
24.  Inflammation, Insulin Resistance, and Diabetes—Mendelian Randomization Using CRP Haplotypes Points Upstream 
PLoS Medicine  2008;5(8):e155.
Background
Raised C-reactive protein (CRP) is a risk factor for type 2 diabetes. According to the Mendelian randomization method, the association is likely to be causal if genetic variants that affect CRP level are associated with markers of diabetes development and diabetes. Our objective was to examine the nature of the association between CRP phenotype and diabetes development using CRP haplotypes as instrumental variables.
Methods and Findings
We genotyped three tagging SNPs (CRP + 2302G > A; CRP + 1444T > C; CRP + 4899T > G) in the CRP gene and measured serum CRP in 5,274 men and women at mean ages 49 and 61 y (Whitehall II Study). Homeostasis model assessment-insulin resistance (HOMA-IR) and hemoglobin A1c (HbA1c) were measured at age 61 y. Diabetes was ascertained by glucose tolerance test and self-report. Common major haplotypes were strongly associated with serum CRP levels, but unrelated to obesity, blood pressure, and socioeconomic position, which may confound the association between CRP and diabetes risk. Serum CRP was associated with these potential confounding factors. After adjustment for age and sex, baseline serum CRP was associated with incident diabetes (hazard ratio = 1.39 [95% confidence interval 1.29–1.51], HOMA-IR, and HbA1c, but the associations were considerably attenuated on adjustment for potential confounding factors. In contrast, CRP haplotypes were not associated with HOMA-IR or HbA1c (p = 0.52–0.92). The associations of CRP with HOMA-IR and HbA1c were all null when examined using instrumental variables analysis, with genetic variants as the instrument for serum CRP. Instrumental variables estimates differed from the directly observed associations (p = 0.007–0.11). Pooled analysis of CRP haplotypes and diabetes in Whitehall II and Northwick Park Heart Study II produced null findings (p = 0.25–0.88). Analyses based on the Wellcome Trust Case Control Consortium (1,923 diabetes cases, 2,932 controls) using three SNPs in tight linkage disequilibrium with our tagging SNPs also demonstrated null associations.
Conclusions
Observed associations between serum CRP and insulin resistance, glycemia, and diabetes are likely to be noncausal. Inflammation may play a causal role via upstream effectors rather than the downstream marker CRP.
Using a Mendelian randomization approach, Eric Brunner and colleagues show that the associations between serum C-reactive protein and insulin resistance, glycemia, and diabetes are likely to be noncausal.
Editors' Summary
Background.
Diabetes—a common, long-term (chronic) disease that causes heart, kidney, nerve, and eye problems and shortens life expectancy—is characterized by high levels of sugar (glucose) in the blood. In people without diabetes, blood sugar levels are controlled by the hormone insulin. Insulin is released by the pancreas after eating and “instructs” insulin-responsive muscle and fat cells to take up the glucose from the bloodstream that is produced by the digestion of food. In the early stages of type 2 diabetes (the commonest type of diabetes), the muscle and fat cells become nonresponsive to insulin (a condition called insulin resistance), and blood sugar levels increase. The pancreas responds by making more insulin—people with insulin resistance have high blood levels of both insulin and glucose. Eventually, however, the insulin-producing cells in the pancreas start to malfunction, insulin secretion decreases, and frank diabetes develops.
Why Was This Study Done?
Globally, about 200 million people have diabetes, but experts believe this number will double by 2030. Ways to prevent or delay the onset of diabetes are, therefore, urgently needed. One major risk factor for insulin resistance and diabetes is being overweight. According to one theory, increased body fat causes mild, chronic tissue inflammation, which leads to insulin resistance. Consistent with this idea, people with higher than normal amounts of the inflammatory protein C-reactive protein (CRP) in their blood have a high risk of developing diabetes. If inflammation does cause diabetes, then drugs that inhibit CRP might prevent diabetes. However, simply measuring CRP and determining whether the people with high levels develop diabetes cannot prove that CRP causes diabetes. Those people with high blood levels of CRP might have other unknown factors in common (confounding factors) that are the real causes of diabetes. In this study, the researchers use “Mendelian randomization” to examine whether increased blood CRP causes diabetes. Some variants of CRP (the gene that encodes CRP) increase the amount of CRP in the blood. Because these variants are inherited randomly, there is no likelihood of confounding factors, and an association between these variants and the development of insulin resistance and diabetes indicates, therefore, that increased CRP levels cause diabetes.
What Did the Researchers Do and Find?
The researchers measured blood CRP levels in more than 5,000 people enrolled in the Whitehall II study, which is investigating factors that affect disease development. They also used the “homeostasis model assessment-insulin resistance” (HOMA-IR) method to estimate insulin sensitivity from blood glucose and insulin measurements, and measured levels of hemoglobin A1c (HbA1c, hemoglobin with sugar attached—a measure of long-term blood sugar control) in these people. Finally, they looked at three “single polynucleotide polymorphisms” (SNPs, single nucleotide changes in a gene's DNA sequence; combinations of SNPs that are inherited as a block are called haplotypes) in CRP in each study participant. Common haplotypes of CRP were related to blood serum CRP levels and, as previously reported, increased blood CRP levels were associated with diabetes and with HOMA-IR and HbA1c values indicative of insulin resistance and poor blood sugar control, respectively. By contrast, CRP haplotypes were not related to HOMA-IR or HbA1c values. Similarly, pooled analysis of CRP haplotypes and diabetes in Whitehall II and another large study on health determinants (the Northwick Park Heart Study II) showed no association between CRP variants and diabetes risk. Finally, data from the Wellcome Trust Case Control Consortium also showed no association between CRP haplotypes and diabetes risk.
What Do These Findings Mean?
Together, these findings suggest that increased blood CRP levels are not responsible for the development of insulin resistance or diabetes, at least in European populations. It may be that there is a causal relationship between CRP levels and diabetes risk in other ethnic populations—further Mendelian randomization studies are needed to discover whether this is the case. For now, though, these findings suggest that drugs targeted against CRP are unlikely to prevent or delay the onset of diabetes. However, they do not discount the possibility that proteins involved earlier in the inflammatory process might cause diabetes and might thus represent good drug targets for diabetes prevention.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050155.
This study is further discussed in a PLoS Medicine Perspective by Bernard Keavney
The MedlinePlus encyclopedia provides information about diabetes and about C-reactive protein (in English and Spanish)
US National Institute of Diabetes and Digestive and Kidney Diseases provides patient information on all aspects of diabetes, including information on insulin resistance (in English and Spanish)
The International Diabetes Federation provides information about diabetes, including information on the global diabetes epidemic
The US Centers for Disease Control and Prevention provides information for the public and professionals on all aspects of diabetes (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050155
PMCID: PMC2504484  PMID: 18700811
25.  P Selectins and immunological profiles in HCV and Schistosoma mansoni induced chronic liver disease 
BMC Gastroenterology  2014;14:132.
Background
Hepatitis C virus (HCV) and Schistosoma mansoni are major causes of chronic liver disease (CLD) in which immune alteration is common. Recent studies suggested that certain platelets and lymphocytes activation markers may have an impact on progression of CLD. This study aimed to evaluate the potential of platelets and lymphocytes activation molecules expression on the pathogenesis of CLD in distinct or concomitant chronic HCV and schistosomiasis mansoni infections.
Methods
The study populations were divided into group-I: patients with chronic schistosomiasis mansoni, group-II: HCV patients without cirrhosis, group-III: patients with combined liver diseases without cirrhosis, group-IV: patients with chronic HCV and liver cirrhosis and group-V: Age and sex matched healthy individuals as normal controls. All groups were subjected to full clinical evaluation, ELISA anti-HCV antibodies screening, parasitological examination for diagnosing S. mansoni and flow cytometry for lymphocyte (CD3, CD4, CD8, CD19, CD22, & CD56) and platelets activation (CD41, CD42 & CD62P (P- selectins)) markers.
Results
The platelet count was significantly decreased in HCV and/or S. mansoni patients. The total T-lymphocytes and T-helper cells were significantly reduced, while T-cytotoxics were increased. The patients possessed a significantly higher platelets activation marker; CD62P (P-selectins) and higher mean fluorescent intensity (MFI) positivity. There were considerable correlations between platelets count and both of CD62P and MFI.
Conclusion
Our Findings suggest an increased expression of certain platelets and lymphocytes activation markers in chronic HCV and S. mansoni induced CLD that may have a role in disease progression.
doi:10.1186/1471-230X-14-132
PMCID: PMC4119237  PMID: 25066324
HCV; Schistosomiasis mansoni; Activated platelets; CD62; Lymphocyte activation

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