Enhanced P-selectin expression and increased soluble CD40 Ligand in patients with Type 1 diabetes mellitus and microangiopathy: evidence for platelet hyperactivity and chronic inflammation

Aims/hypothesis Platelet activation, endothelial dysfunction and inflammation may be involved in early stages of diabetic microangiopathy. We therefore investigated patients with Type 1 diabetes mellitus, without (n=19) and with (n=20) microangiopathy, matched for glycaemic control and duration of disease, and matched with healthy control subjects (n=27).Methods Platelet activation was measured as platelet P-selectin expression using whole blood flow cytometry and as soluble P-selectin by immunoassay. Von Willebrand factor antigen in plasma, serum soluble E-selectin, CD40 ligand (sCD40L) and C-reactive protein (CRP) served as markers for endothelial function and inflammation.Results Thrombin-induced platelet P-selectin expression was enhanced, and soluble P-selectin and sCD40L concentrations were increased in patients with microangiopathy compared with the control subjects (p<0.01 for both) and with patients without microangiopathy (p<0.05 for P-selectin expression and sP-selectin), whereas all three parameters were similar in patients without microangiopathy and in the control subjects. CRP and soluble E-selectin were increased in patients with microangiopathy, compared with the control subjects (p<0.01 and p<0.05), whereas von Willebrand factor did not differ between the groups.Conclusions/interpretation Microangiopathy in Type 1 diabetes is associated with platelet hyperactivity, endothelial dysfunction and low-grade inflammation, indicating an increased risk for cardiovascular disease.Abbreviations sP-selectin Soluble P-selectin - sCD40L soluble CD40 Ligand - CRP C-reactive protein     

Inverse relationship between erythrocyte size and platelet reactivity in elderly

Previous work indicates that erythrocytes (RBCs) accumulate β-amyloid X-40 (Aβ₄₀) in individuals with Alzheimer disease (AD) and to a lesser extent in healthy elderly. The toxin damages RBCs and increases their mean corpuscular volume (MCV). Furthermore, AD platelets demonstrate lower reactivity. This study investigated interactions between RBCs and platelets. Older individuals with moderate hypertension (n = 57) were classified into two groups, depending on MCV in whole blood: The MCV^high group comprised individuals with higher MCV (n = 27; 97 ± 3(SD) fl) and MCV^low group had relatively lower MCV (n = 30; 90 ± 3(SD) fl). Flow cytometry was used to determine platelet reactivity, i.e., the surface binding of fibrinogen after provocation. Adenosine diphosphate (ADP) and a thrombin receptor-activating protein (TRAP-6) were used as agonists. Subsequently, blood cells were divided according to density into 17 subfractions. Intra-RBC Aβ₄₀ content was analyzed and in all platelet populations surface-bound fibrinogen was determined to estimate platelet in vivo activity. We found Aβ₄₀ inside RBCs of approximately 50% of participants, but the toxin did not affect MCV and platelet reactivity. In contrast, MCV associated inversely with platelet reactivity as judged from surface-attached fibrinogen after ADP (1.7 μmol/L) (p < 0.05) and TRAP-6 provocation (57 μmol/L (p = 0.01) and 74 μmol/L (p < 0.05)). In several density fractions (nos. 3, 4, 8, 11–13 (p < 0.05) and nos. 5–7 (p < 0.01)) MCV linked inversely with platelet-attached fibrinogen. In our community-dwelling sample, enhanced MCV associated with decreased platelet reactivity and lower in vivo platelet activity. It resembles RBCs and platelet behavior in AD-type dementia.     

Gender,race and diet affect platelet function tests in normal subjects,contributing to a high rate of abnormal results

To assess sources of variability in platelet function tests in normal subjects, 64 healthy young adults were tested on 2–6 occasions at 2 week intervals using four methods: platelet aggregation (AGG) in platelet‐rich plasma (PRP) in the Bio/Data PAP‐4 Aggregometer (BD) and Chrono‐Log Lumi‐Aggregometer (CL); and AGG in whole blood (WB) in the CL and Multiplate Platelet Function Analyser (MP), with ATP release (REL) in CL‐PRP and CL‐WB. Food and medication exposures were recorded prospectively for 2 weeks prior to each blood draw. At least one AGG abnormality was seen in 21% of 81 drug‐free specimens with CL‐PRP, 15% with CL‐WB, 13% with BD‐PRP and 6% with MP‐WB, increasing with inclusion of REL to 28% for CL‐PRP and 30% for CL‐WB. Epinephrine AGG and REL were significantly reduced in males (P < 0·0001). Ristocetin AGG and collagen and thrombin REL were significantly reduced in Blacks (P < 0·0001). One‐third of specimens drawn following flavonoid‐rich food exposures had aberrant results, compared to 8·5% of specimens without such exposures (P = 0·0035). PRP tests had less intra‐individual variation than WB tests. Gender, race, diet and test system affected results of platelet function testing in healthy subjects, suggesting caution when interpreting the results of platelet function testing in patients.     

Lipid Composition of Platelets in Patients with Non-insulin-dependent Diabetes Mellitus: Studies Before and After Treatment of Diabetes

The study was designed to investigate whether impaired composition of platelet lipids in untreated diabetic patients improved after diabetic treatment. Fourteen untreated patients with non-insulin-dependent diabetes mellitus (NIDDM) and 15 healthy control subjects were studied. In the diabetic patients, the ratio of free cholesterol to phospholipid (FC/PL) in platelets of 0.33 ± 0.02 (mean ± SEM) at pre-treatment, which was statistically (p < 0.05) higher than that of 0.26 ± 0.02 in control subjects, was significantly decreased to the value of 0.29 ± 0.02 (p < 0.01) after insulin therapy. Platelet FC level of 9.77 ± 0.77 μg 10^?8 cells pre-treatment was significantly (p < 0.01) reduced to the value of 7.72 ± 0.38 μg 10^?8 cells post-treatment. Platelet PL level showed no significant changes after the treatment. There was a significantly (p < 0.01) positive correlation between the decrease in FC/PL of platelets and that in haemoglobin A_1c (HbA_1c) after treatment for diabetes (rs = ?0.729). These results indicate that the impaired lipid composition in platelets can be improved after an adequate glycaemic control in patients with NIDDM.     

Influence of vitamin E on the antiplatelet effect of acetylsalicylic acid in human blood

We analysed the in vitro interaction between acetylsalicylic acid and vitamin E on the principal antiplatelet sites of action of acetylsalicylic acid, i.e., platelet aggregation, prostanoid production in platelets and leukocytes, and nitric oxide synthesis. Aggregation was measured in whole blood and in platelet-rich plasma (PRP) with ADP, collagen or arachidonic acid as platelet inducers, and we measured the production of thromboxane B₂, prostacyclin and nitric oxide. Vitamin E potentiated the antiplatelet effect of acetylsalicylic acid in both whole blood and PRP. In PRP induced with collagen the IC₅₀ for acetylsalicylic acid alone was 339?±?11.26, and that of acetylsalicylic acid?+?vitamin E was 0.89?±?0.09 (P?<?0.05). Vitamin E did not enhance inhibition of platelet thromboxane production by acetylsalicylic acid. Vitamin E spared or even increased prostacyclin levels, and acetylsalicylic acid?+?vitamin E diminished the inhibition of prostacyclin synthesis by acetylsalicylic acid (IC₅₀ acetylsalicylic acid alone?=?1.81?±?0.15?µM; IC₅₀ acetylsalicylic acid?+?vitamin E?= 12.92?±?1.10?µM, P?<?0.05). Vitamin E increased the effect of acetylsalicylic acid on neutrophil nitric oxide production 42-fold (P?<?0.05). We conclude that vitamin E potentiates the antiplatelet effect of acetylsalicylic acid in vitro, and thus merits further research in ex vivo studies.     

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2?×?10³?leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10?µM) and ATP (10–100?µM) was markedly reduced, but to ATP (1000?µM) was enhanced (all p?<?0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t_1/2?=?4?min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4?min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.     

Protein A immunoadsorption in chronic refractory ITP reverses increased platelet activation but fails to achieve sustained clinical benefit

Adults with chronic relapsing ITP present a difficult therapeutic challenge. The ongoing antibody- mediated platelet destruction in this group might be expected to be associated with increased expression of platelet surface membrane activation antigens. We have studied a group of 10 patients with refractory ITP and 35 healthy controls. Using an immediate, sensitive, unfixed, whole blood, flow cytometric method to detect platelet surface P-selectin and GP53, we have detected markedly increased platelet activation in the ITP group compared with the controls (P-selectin; patient median 24.5% v control median 2.0%, GP53 median 6.5% v 2.1%, P < 0.01 for both). Five patients underwent protein A immunoadsorption therapy. The effect of protein A immunoadsorption on platelet activation before, during and after 18 treatments in these patients was studied and patients were followed-up to assess clinical outcome. Platelet-associated immunoglobulin measurements were made before and at the end of six treatments. Platelet activation decreased after immunoadsorption. P-selectin expression fell significantly; pre- and post-treatment median values differed by 15.5%, P < 0.01, for GP53 the difference was 2.5.%, P = NS. A reduction in both platelet-associated IgG (median reduction of 11.8 ng/10⁶ platelets, P = 0.08) and IgM (7.6 ng/10⁶ platelets, P = 0.06) was recorded.     

Effect of thrombopoietin-receptor agonists on circulating cytokine and chemokine levels in patients with primary immune thrombocytopenia (ITP)

Background: Thrombopoietin-receptor-agonists (TPO-RAs) increase platelet production in Immune Thrombocytopenia (ITP) by stimulating Mpl. The effect of TPO-RAs on inflammatory cytokine production in ITP patients has not been well investigated. Methods: Plasma samples from 48 ITP patients treated with TPO-RAs (median age 50 years (inter-quartile range; IQR 20–69), median platelet counts 24 × 10⁹/L (IQR 15–47 × 10⁹/L), 28 females) and 16 healthy controls (nine females, median age 37 years, IQR 22–51 years) were collected before and during treatment, and analyzed for a panel of cytokines and chemokines by enzyme-linked immunosorbent assay and immuno-bead-based multiplex assay. Results: Elevated levels of C-X-C motif chemokine 10 (CXCL10; p < 0.001) and osteoprotegerin (OPG; p < 0.05) were observed in pretreatment samples compared to controls; these levels decreased during 6 months of treatment. Pretreatment levels of transforming growth factor (TGF)-β were lower than in healthy controls and increased after 6 months of treatment (p < 0.05). Levels of sCD40L increased after 6 months of treatment (p < 0.05), but decreased thereafter to pretreatment values. The increase in TGF-β and sCD40L may reflect increased platelet turnover. Levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-10 did not change during treatment. Conclusion: These findings suggest that treatment with TPO-RA creates a more balanced steady-state of immune activation.     

The influence of Rubus idaeus and Rubus caesius leaf extracts on platelet aggregation in whole blood. Cross-talk of platelets and neutrophils

Recently, polyphenols have gained attention as potential natural cardioprotective therapeutics, due to their antiplatelet, anti-inflammatory and anticoagulant activity. Species belonging to the genus Rubus sp. have been reported to be a source of polyphenolic compounds with antioxidative proprieties and beneficial biological activities. This study investigates the effects of leaf extracts obtained from red raspberry (Rubus idaeus L.) and European dewberry (Rubus caesius L.) on the reactivity of blood platelets.

In ADP-stimulated blood, raspberry and dewberry extracts (15 µg/ml) markedly decreased platelet surface membrane expression of activated GPIIbIIIa receptor by 16% and 21%, respectively (P < 0.01) and significantly inhibited platelet aggregation (by 31–41% for raspberry and by 38–55% for dewberry, P < 0.01). In platelet-rich plasma (PRP), the extracts had no effect on ADP-induced platelet aggregation.

The effectiveness of the extracts in whole blood and the lack of their activity in PRP indicate that leukocytes are likely to participate in the platelet response to the extracts. Our experiments show that the extracts significantly reduced the amount of free radicals released by activated neutrophils in whole blood (P < 0.001), as well as in suspensions of isolated neutrophils (P < 0.05). Moreover, the reduced number of neutrophils leads to the decreased efficiency of the extracts in the inhibition of platelet aggregation. In summary, our findings show that the raspberry and dewberry leaf extracts considerably modulated blood platelet reactivity in whole blood: they influenced blood platelet aggregation, possibly via the modulation of the redox status dependent on the oxidative activity of neutrophils.     

Glycocalicin in the diagnosis and management of immune thrombocytopenia

Abstract: We studied glycocalicin (GC), expressed as plasma GC concentration and as GC index (ratio to platelet count), in 129 thrombocytopenic patients (platelet count <100×10⁹/1) and 60 sex- and age-matched controls. Seventy-two patients had idiopathic immune thrombocytopenia, 32 secondary immune thrombocytopenia, 8 microangiopathic thrombocytopenia and 17 thrombocytopenia secondary to bone marrow aplasia. Patients with immune thrombocytopenia (ITP) were also subclassified, according to their clinical behaviour, as having active disease or being in spontaneous or therapy-induced partial remission. A significant correlation was found between glycocalicin levels and platelet count both in normals and in patients with bone marrow aplasia (r = 0.75). ITP patients showed a GC index significantly higher than controls (6.02±7.87 vs. 0.9±0.2, p<0.001). When ITP patients with similar platelet count (30–50×10⁹/l) were studied, the mean level of GC and the GC index were significantly higher in those patients with active disease than in those in remission (0.97±0.38 vs. 0.58±0.17 μg/ml, p<0.05; 6.41±2.64 vs. 3.44±0.94, p<0.05, respectively). A longitudinal study performed in 10 patients with different subtypes of ITP suggested a positive correlation between GC index and the activity of the disease. The GC value and GC index were significantly higher in patients with microangiopathic thrombocytopenia than in controls (1.44±0.73 vs. 0.8±0.16 μg/ml, p<0.01; and 18.77±22.23 vs. 0.9±0.2, p<0.001, respectively). The GC value was significantly lower in bone marrow failure (0.15±0.04 μg/ml, p<0.01) compared to controls, while no difference was observed in the GC index. Our data confirm that the GC index is helpful in differentiating thrombocytopenia due to increased platelet destruction from the one due to impaired production. In addition, the assay has been proven useful in the differential diagnosis of different ITP subtypes and their follow-up.     

Evaluation of the immature platelet fraction in the diagnosis and prognosis of childhood immune thrombocytopenia

Rapid assessment of platelet production would distinguish between thrombocytopenia due to decreased platelet production or increased peripheral platelet destruction. We evaluated the value of immature platelet fraction (IPF) in differentiating immune thrombocytopenia (ITP) from thrombocytopenia secondary to bone marrow failure and its potential use as a prognostic marker. Forty-one young patients with ITP were compared with 14 patients with hematological malignancies under chemotherapy, representing a control group with thrombocytopenia due to bone marrow suppression and 30 age- and sex-matched healthy controls. Patients were studied stressing on bleeding manifestations, organomegaly/lymphadenopathy and therapy. Complete blood count including IPF was performed using Sysmex XE-2100. ITP patients were classified into two subgroups: acute ITP with spontaneous resolution within 3 months from diagnosis and chronic ITP that lasted ≥1 year from diagnosis. Median IPF was 11.8% in patients with ITP, 7% in those with hematological malignancy and 3% in the control group (p?<?0.001). ITP patients had significantly higher mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR) and IPF compared with patients with malignancy or healthy controls, while plateletcrit (PCT) was significantly lower in ITP patients than other groups (p?<?0.001). IPF was increased in patients with chronic ITP compared with acute ITP group (p?<?0.001). Patients with active ITP had the highest IPF followed by those in partial remission, while ITP patients in remission had the lowest IPF. IPF was positively correlated to the number of lines of treatment used, MPV, PDW and P-LCR, while negatively correlated to platelet count and PCT among ITP patients (p?<?0.001). Multiple regression analysis showed that platelet count and P-LCR were independently related to IPF. ROC curve analysis revealed that the cut-off value of IPF at 9.4% could be diagnostic for ITP patients with a sensitivity of 88% and a specificity of 85.7%. We suggest that IPF may be a rapid and inexpensive automated marker for etiology of thrombocytopenia and can be integrated as a standard parameter to evaluate the thrombopoietic state of the bone marrow. It may be considered as a potential prognostic marker for the development of chronic ITP.     

Increased β-Thromboglobulin in Essential Hypertension: Interactions between Arterial Plasma Adrenaline,Platelet Function and Blood Lipids

ABSTRACT Twenty-three 50-year-old men with untreated, essential hypertension had elevated plasma concentrations of the platelet release product β-thromboglobulin (BTG) compared to 14 age-matched control men (p<0.01). BTG correlated with arterial plasma adrenaline concentrations in the hypertensive (r=0.44, p<0.05), normotensive (r=0.73, p<0.01) and combined group (r=0.51, p<0.01). Significant correlations (p<0.05) between BTG and cholesterol (LDL + VLDL fraction) were observed both in the hypertensive and the normotensive group. In the hypertensive group arterial adrenaline correlated with cholesterol (LDL + VLDL) (p<0.05). These findings are consistent with increased platelet activity in middle-aged men with essential hypertension, and may indicate that plasma adrenaline influences platelet function. The risk factors for coronary artery disease (blood pressure, lipid status, stress as evidenced by catecholamine release and platelet function) were positively related. Measurement of arterial instead of venous adrenaline is essential for the demonstration of the associations presented.     

Of Mice and Men: Neuropeptide Y and Its Receptors Are Associated with Atherosclerotic Lesion Burden and Vulnerability

Neuropeptide Y (NPY), a sympathetic and platelet-derived vascular mitogen and angiogenic factor, has been implicated in atherosclerosis in animal and human genetic studies. Here we evaluate its association with human and murine atherosclerosis, and assess the role of platelet-derived NPY in lesion vulnerability. NPY immunoreactivity (NPY-ir) was measured in the platelet-poor and platelet-rich (PRP) plasmas, and NPY receptors (mitogenic Y1R and angiogenic Y2 and Y5Rs), CD26/DPPIV (a protease forming Y2/Y5-selective agonist), CD31-positive vascularity, and lesion morphology assessed by histo- and immunocyto-chemistry—in patients with peripheral artery disease (PAD) and healthy volunteers, and in lard-fed ApoE−/− mice. NPY and NPY-R immunostaining was greater in lesions from PAD patients compared to normal vessels of healthy volunteers (p < 0.001), and localized to smooth muscle cells, macrophages, and adventitial/neovascular endothelial cells. CD26/DPPIV staining co-localized with CD31-positive endothelial cells only in atherosclerotic lesions. NPY-ir in PRP (but not plasma) and vascular immunostaining was higher (p < 0.05 and 0.001, respectively) in men (not women) with PAD compared to healthy subjects. A similar gender specificity was observed in mice. PRP NPY-ir levels correlated with lesion area (p = 0.03), necrotic core area, and the necrotic core-to-lesion area ratio (p < 0.01) in male, but not female, mice. Also males with neovascularized lesions had higher PRP NPY-ir levels than those lacking lesion microvessels (p < 0.05). NPY and its Rs are up-regulated in human and murine atherosclerotic lesions suggesting pathogenic role. DPPIV expression by microvascular endothelium in atherosclerotic tissue may shift NPY’s affinity toward angiogenic Y2/Y5Rs, and thus enhance angiogenesis and lesion vulnerability. Remarkably, plaque neovascularization was associated with increased NPY-ir in PRP in males but not females, suggesting that platelet NPY may be a novel mediator/marker of lesion vulnerability particularly in males, for reasons that remain to be determined. Both animal and human data suggest that NPY is an important contributor to, and platelet NPY-ir a marker of, atherosclerotic lesion burden and vulnerability but only in males, perhaps due to androgen-dependent up-regulation of NPY, previously shown in rats.     

Lower expression of PD-1 and PD-L1 in peripheral blood from patients with chronic ITP

Background: T-cell dysregulation is a major event involved in immune thrombocytopenic purpura (ITP). Increasing data have indicated that abnormal expression of T-cell immunosuppressive receptors, such as programmed death (PD) 1 and cytotoxic T lymphocyte antigen-4 (CTLA-4), may be related to autoimmune disease pathogenesis.

Methods: We analyzed the expression levels of PD-1, its ligand PD-L1, and CTLA-4 in peripheral blood mononuclear cells from 18 patients with chronic ITP by real-time polymerase chain reaction, and samples from 20 healthy individuals served as control.

Results: The results demonstrated significantly lower expression of PD-1 (median: 0.0015) and PD-L1 (median: 0.0572) in chronic ITP patients compared with healthy individuals (PD-1: median: 0.0117, P?<?0.0001; PD-L1: median: 0.5428, P?<?0.0001), while there was no significant difference in the CTLA-4 expression level between the chronic ITP patients (median: 0.0818) and healthy individuals (median: 0.1667) (P?=?0.219). Moreover, a positive correlation between the expression levels of PD-1 and PD-L1 (r_s?=?0.486, P?=?0.041) and CTLA-4 and PD-1 (r_s?=?0.643, P?=?0.004) in the chronic ITP patients was found.

Conclusion: Consistently lower expression of T-cell immunosuppressive receptors is a common characteristic of chronic ITP, which may be associated with its pathogenesis.     

Reduced clot retraction rate and altered platelet energy production in patients with asthma

Objective: Asthma enhances the risk of pulmonary embolism. The mechanism of this phenomenon is unclear. Methods: We evaluated the kinetics of clot formation, clot retraction rate (CRR), clot volume at 40 min, the rate of lactate production (a marker of aerobic glycolysis in platelets in contracting clots), blood eosinophil count (EOS), nitric oxide in exhaled breath (FE_NO), and spirometry (FEV₁) in 50 healthy controls and in 81 allergic asthmatics (41 subjects with steroid-naïve asthma and 40 with steroid-treated asthma). Results: Thromboelastometry revealed that only steroid-treated asthmatics had slightly activated coagulation. Compared with healthy controls, whole asthmatics demonstrated (p < 0.05) reduced CRR, higher clot volume at 40 minutes, higher FE_NO, decreased FEV₁, elevated EOS, and augmented lactate production in retracting clots. Reduced CRR was observed also in the absence of native plasma. In whole study population (asthmatics and healthy controls), CRR positively correlated with spirometry (r_S = 0.668, p = <0.001) and negatively with FE_NO (r_S = ?0.543; p < 0.001), EOS (r_S = ?0.367, p < 0.002), and lactate production (r_S = ?0.791; p < 0.001). However, in steroid-treated asthmatics, the CRR did not correlate with FE_NO and EOS. In all study patients lactate production negatively correlated with FEV₁ and positively with FE_NO. Conclusion: Collectively, this data is consistent with the hypothesis that, in asthmatics, reactive nitrogen species produced in the lungs may reduce platelet contractility (and CRR) through the diminution of platelet energy production. CRR inhibition would predispose asthmatics to pulmonary embolism.     

Platelet Counts in Myocardial Infarction,Angina Pectoris and Peripheral Artery Disease

ABSTRACT Serial determinations of peripheral venous platelet counts were performed in 43 consecutive patients with acute chest pain. On admission, patients with acute myocardial infarction (AMI) had a significantly lower mean platelet count (p<0.05) than patients without AMI, whereas hematocrit was higher (p<0.025). Thereafter a further reduction was seen with steadily reduced platelet counts by about 20%, both in comparison with healthy controls (p<0.001) (n=113) and patients with peripheral artery disease (PAD) without heart symptoms (p<0.005) (n=54). The platelet number increased one week after admission and even thrombocytosis was observed. The changes in platelet number during AMI seem to parallel the changes in platelet function. Patients with PAD had normal mean platelet counts. Female patients as well as healthy subjects had significantly higher values than men (p<0.01).     

Reproducibility and standardized reporting of the vasodilator-stimulated phosphoprotein phosphorylation assay

The platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation test is a new platelet function assay used to determine responsiveness to clopidogrel. We investigated if a commercially available VASP assay has a good reproducibility when conducted with half or quarter of the recommended volume of reagents. Secondly, we hypothesized that the platelet reactivity indexes (PRI) in the VASP assay, calculated with three possible measurements of fluorescence intensities (FI): mean, geometric mean or median, are not equal. The VASP assay was performed in 280 patients on clopidogrel therapy and 35 volunteers without clopidogrel therapy. The VASP assay had an excellent reproducibility even if half or quarter of the volume of reagents recommended by the manufacturer were used: there was a high correlation between analyses with full vs. half and full vs. quarter volumes of reagents (r = 0.94; r = 0.93; p < 0.001, respectively). The PRI index was 4% higher when calculated with the geometric mean FI or the median FI (83%) as compared to the mean FI (80%; p < 0.001) in healthy volunteers without clopidogrel therapy. The PRI was 6% higher when calculated with the geometric mean FI or the median FI (52%) as compared to the mean FI (49%; p < 0.01) in patients on clopidogrel therapy. In conclusion, using half of the volume of reagents may constitute a cost-saving strategy for any laboratory performing the assay. Secondly, differences in calculation of PRI (mean, geometric mean or median fluorescence intensities) significantly contribute to the variability of results and reported cut-off values of the PRI. This variability can be easily avoided to facilitate comparison of data between different laboratories.     

Changes in platelet function with inflammation in patients undergoing vascular surgery

The role of platelets in ischaemic events is well established. Aspirin represents the default antiplatelet and blocks the metabolism of arachidonic acid (AA) at the cyclo-oxygenase enzyme (COX). AA is commonly used as a test of response to aspirin, but recent data raise uncertainty about the validity of this approach. Specifically, in some patients AA-induced clotting is not suppressed, but the level of COX-dependent AA metabolite, thromboxane B2 (TXB₂) is negligible. Furthermore, AA-induced whole blood clotting varies dynamically in individuals, who are aspirin responsive according to TXB₂ levels.

The aim of this study was to assess the level of AA-, ADP- and thrombin-mediated platelet reactivity in patients on aspirin before, during, and after major vascular surgery, which represents a model of on/off vascular inflammation. Firstly, we hypothesized, that in association with this inflammatory episode AA-, ADP- and thrombin-induced clotting would change in a dynamic manner. Secondly, that AA-induced clotting will be modified despite complete suppression of platelet TXB₂ production by aspirin throughout the periprocedural period, possibly via a lipoxygenase-mediated mechanism.

Fourty patients underwent major vascular surgery (open abdominal aortic aneurysm operation, infrainguinal bypass for subcritical limb ischaemia or peripheral aneurysm repair with bypass). They were all on 75 mg of aspirin prior to and throughout the perioperative period and received 5000 units of unfractionated heparin intraoperatively. AA-, ADP-, and thrombin-induced clotting, AA metabolites (TXB₂ and 12-Hyroxyeicosatetraenoic acid (12-HETE)) and inflammatory markers (CRP, IL-6, TNF-α and CD40) were measured pre-procedure and at 2, 24, 48 hours, 3 to 5 days and 3 months after surgery. AA-, ADP- and thrombin-induced platelet reactivity was assessed using thrombelastography. TXB₂, 12-HETE, IL-6, TNF-α, CD40 were determined using the sequential competitive binding Enzyme-Linked ImmunoAssay technique and CRP was determined using an immune-turbidimetric test on human serum.

There was a transient rise in inflammatory markers in the early perioperative period (CRP at 24, 48 hours and 3 to 5 days p < 0.001 and IL-6 at 2, 24, 48 hours and 3 to 5 days p < 0.001 as compared to baseline). Patients had negligible levels of TXB₂ throughout, confirming a consistent therapeutic response to aspirin. There was a transient rise in thrombin-mediated clotting (MA_Thrombin at 48 hours p = 0.001 and 3 to 5 days p < 0.001) and a fall in AA- and ADP-induced clotting in the early post op period (both MA_AA and MA_ADP p = 0.001 at 2 hours). At 3 months, the level of AA- and ADP-induced clotting was significantly higher than at baseline (p = 0.008 for MA_AA and p = 0.002 for MA_ADP), hence demonstrating a rebound effect.

These data demonstrate a novel dynamic variation in platelet aggregation with acute vascular inflammation, including AA-induced whole blood clotting which is apparently COX-1 independent.     

Platelet membrane lipid fluidity and intraplatelet calcium mobilization in type 2 diabetes mellitus

Abstract: The aim of the present study was to relate the impairments in calcium mobilization and/or release to the altered membrane dynamics in platelets from patients with type 2 diabetes mellitus. Higher expression of P-selectin (1.4-fold, NS) and the reduction in GPIbα expression (by 27.8 ± 16.7%, p < 0.0002), as well as the increased fractions of platelet microparticles (p < 0.03), reflected more intensified platelet release reaction in diabetic platelets. Overall, diabetic platelets appeared more vulnerable to stimuli facilitating calcium mobilization (by 41%, p < 0.01) and less susceptible to preventive effects of the agents hampering calcium release from intraplatelet storage pools (by 38%, p < 0.01). Both the increased calcium mobilization from intraplatelet storage pools and higher levels of intracellular free calcium in the presence of procaine in diabetic platelets correlated with the reduced platelet membrane lipid fluidity (resp. p_R < 0.03 and p_R < 0.015). We conclude that the biophysical state of platelet membrane components in diabetes mellitus is the crucial determinant of platelet hyperfunction and probably contributes to the intensified calcium mobilization in diabetic platelets. The depressed preventive effects of procaine on platelet release reaction and calcium mobilization in diabetic platelets may result from the primary dislocations and/or distortions of membrane components caused by the diabetic state.     

Retrospective view of primary Raynaud's phenomenon in childhood

ObjectivesPrimary Raynaud's phenomenon (PRP) manifests as episodes of transient spasms of peripheral blood vessels. To elucidate the clinical clues and laboratory characteristics will facilitate the identification of PRP.MethodsA retrospective data collection of clinical and laboratory characteristics of 58 children with PRP was performed between January 2007 and December 2016.ResultsA positive ANA test at lower titers <1:100 was detected in 24.1% of the patients. There was a significant relationship between presence of ANA positivity and migraine in female patients with PRP (p = 0.01; p = 0.020 respectively). The most common accompanying disorder was migraine which was detected in 37.9% of all patients with PRP. Hemoglobin and serum ferritin levels were significantly lower in PRP patients with migraine (p = 0.045; p < 0.05, respectively). Additionally, the mean platelet volume (MPV) measurements were significantly higher in patients with migraine compared to those without migraine (p = 0.045; p < 0.05 respectively).DiscussionThere is limited data concerning childhood PRP. For the first time we showed a high frequency of migraine in childhood PRP. Anemia and high MPV could be the underlying triggering factors of these two episodic diseases.