Giuseppe Guglielmini

Acute, short-term hyperglycemia enhances shear stress-induced platelet activation in patients with type II diabetes mellitus

OBJECTIVES The aim of our study was to assess whether acute, short-term hyperglycemia affects platelet reactivity in patients with Type II diabetes mellitus (T2DM). BACKGROUND Hyperglycemic spikes are thought to precipitate ischemic events in T2DM. Previous studies have shown in vivo platelet activation in diabetes; however, no studies have assessed whether acute in vivo hyperglycemia induces further activation of platelets. METHODS In a cross-over, randomized, double-blind study, 12 patients with T2DM underwent 4 h of either acute hyperglycemia (13.9 mmol/l, 250 mg/dl) or euglycemia (5.5 mmol/l, 100 mg/dl). Shear stress-induced platelet activation, P-selectin and lysosomal integral membrane protein (LIMP) expression on platelets in the bleeding-time blood, urinary 11-dehydro-thromboxane B(2) (TxB(2)) excretion, von Willebrand factor:antigen (vWF:Ag), and von Willebrand factor:activity (vWF:activity) were measured before and after hyperglycemia or euglycemia. RESULTS Shear stress-induced platelet activation, P-selectin and LIMP expression on platelets in the bleeding-time blood, and urinary 11-dehydro-TxB(2) excretion increased significantly after hyperglycemic clamping, whereas no changes were observed after euglycemic clamping. Plasma vWF:Ag and vWF:activity increased strikingly in parallel fashion after hyperglycemic clamping, whereas no changes were observed after euglycemic clamping. CONCLUSIONS Our data demonstrate that acute, short-term hyperglycemia induces an increased activation of platelets exposed to high shear stress conditions in vitro (filtration method) or in vivo (bleeding time). In vivo platelet activation is reflected by an increased urinary excretion of 11-dehydro-TxB(2). The increased levels of vWF in the circulation correlate with the increase in platelet activation markers and may indicate some degree of causation. Acute, short-term hyperglycemia in T2DM may precipitate vascular occlusions by facilitating platelet activation.

Effects of resveratrol and other wine polyphenols on vascular function: An update

Several epidemiologic observations show that moderate wine drinking reduces cardiovascular morbidity and mortality. Wine contains several polyphenols, and among them, resveratrol in particular has been shown to exert a number of important biologic activities on the cardiovascular system that may contribute to the protective effects of wine. The mechanisms through which resveratrol and other wine polyphenols protect from ischemic cardiovascular events are many, but protection from oxidative stress and radical oxygen species production, a facilitating activity on nitric oxide production and activity and the ability to modulate the expression of adhesive molecules by blood cells and the vascular wall seem to be the most important. In this overview, the in vitro and in vivo evidence on the activity of resveratrol on vascular function and circulating blood cells, with a special emphasis on blood platelets, is thoroughly presented.

HIV type 1 infection, and not short-term HAART, induces endothelial dysfunction.

Objective:Ischemic cardiovascular events increasingly occur during long-lasting HIV infection and are attributed either to the infection itself or to the use of HAART. Endothelial dysfunction and platelet activation are markers of atherosclerosis. Our aim was to assess whether patients with chronic HIV infection present endothelial dysfunction and whether this is the consequence of infection or of HAART. Design:Fifty-six HIV-infected patients were studied in a retrospective cohort study before and 3, 6, 12 and 24 months after starting HAART with protease inhibitors (n = 28) or nonnucleoside reverse transcriptase inhibitors (n = 28), and compared with 28 age-matched and sex-matched healthy controls, and with 10 naive HIV-infected patients studied at diagnosis and after 12 months of untreated infection. Methods:Soluble endothelial and platelet activation markers were measured in plasma by flow cytometry. Results:Soluble P-selectin, soluble vascular cell adhesion molecule-1, monocyte chemoattractant protein-1 and von Willebrand factor were significantly higher in HIV-infected patients than in healthy controls, whereas soluble CD40 ligand and tissue type plasminogen activator were within normal range. During follow-up, soluble vascular cell adhesion molecule-1, monocyte chemoattractant protein-1 and von Willebrand factor but not soluble P-selectin decreased progressively, without significant differences between protease inhibitors and nonnucleoside reverse transcriptase inhibitors treatment. In naive, untreated patients, increased plasma markers of endothelial dysfunction were confirmed at diagnosis, with no changes upon follow-up. Conclusion:Chronic HIV infection, and not its pharmacological treatment, induces alterations of markers of endothelial function. Short-term treatment with HAART reduces some markers of endothelial dysfunction, with no differences between protease inhibitors and nonnucleoside reverse transcriptase inhibitors.

Intraplatelet signaling mechanisms of the priming effect of matrix metalloproteinase‐2 on platelet aggregation

Summary.  Objective: Platelets contain and release some matrix metalloproteinases (MMPs), enzymes involved in the degradation of extracellular matrix, and one of these (MMP‐2) exerts a proaggregatory effect. We explored the signal transduction mechanisms activated by MMP‐2 in human blood platelets. Methods and results:Recombinant, human MMP‐2, added before stimulation with subthreshold doses of different agonists, potentiated platelet activation, calcium influx, IP3 formation, and pleckstrin phosphorylation. Wortmannin and LY29400, two PI3‐K inhibitors, suppressed the potentiating effects of MMP‐2 and preincubation with MMP‐2 enhanced the thrombin‐induced association of the p85α PI3‐K subunit with the cytoskeleton and increased the phosphorylation of PKB. Protein tyrosine kinase inhibitors, MAP kinase inhibitors, PLA2 inhibitors, cyclooxygenase inhibitors and antagonists of the P2Y1 and P2Y12 receptors did not affect the potentiating activity of MMP‐2 on platelets. Conclusion: Our data show that MMP‐2, at a concentration released by activated platelets, facilitates platelet activation acting at the level of a second messenger system common to different agonists and related to the activation of PI3‐K. Platelet‐released MMP‐2 may contribute to platelet activation in vivo.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression1

Summary.  Background: NO‐releasing statins are new chemical entities, combining HMG‐CoA reductase inhibition and slow NO release, that possess stronger anti‐inflammatory and antiproliferative activities than the native statins. Objective: We evaluated the antithrombotic effects of nitropravastatin (NCX‐6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. Methods and results: In vitro, NCX‐6550 inhibited (1) U46619‐ and collagen‐induced platelet aggregation in buffer and plasma; (2) collagen‐induced P‐selectin expression in whole blood and (3) platelet adhesion to collagen‐coated coverslips under high shear stress. These effects were displayed at concentrations of NCX‐6550 ranging from 25 to 100 μm, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 μm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS‐ and PMA‐induced TF expression by blood mononuclear cells was also inhibited by NCX‐6550 (IC50 13 μm), but not by pravastatin, as assessed by functional and immunological assays and by real‐time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX‐6550 (24–48 mg kg−1) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. Conclusions: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Platelets release matrix metalloproteinase-2 in the coronary circulation of patients with acute coronary syndromes: possible role in sustained platelet activation

AIMS To investigate whether selected matrix metalloproteinases (MMPs) are released in the coronary circulation of patients with acute coronary syndrome (ACS), whether this release is related to platelet activation, and whether it contributes to sustained platelet activation. METHODS AND RESULTS Blood from the aorta (Ao) and the coronary sinus (Cs) was obtained from 21 controls (non-cardiac chest pain), 24 stable angina (SA), and 30 ACS patients, before performing percutaneous transluminal coronary angioplasty. Selected MMPs, some platelet activation- and atheroma-related markers, and the platelet activation-potentiating activity of plasma were measured. Total MMP-2, active MMP-2, and MMP-9 were released in the coronary circulation of patients with ACS, but not of those with SA or controls. Similarly, transcoronary gradients of β-thromboglobulin (β-TG) and platelet factor 4, two platelet-specific proteins, and of soluble CD40L and secretory phospholipase A₂ (sPLA₂), markers of inflammation and platelet activation, were higher in ACS patients than in the other groups. In contrast, plasma monocyte chemoattractant protein-1, a platelet-unrelated marker of atherogenesis, was not increased in the Cs compared with Ao in any of the groups. Transcoronary gradients of both β-TG and sPLA₂ correlated with those of total and active MMP-2 in ACS, but not in controls or SA. Plasma from the Cs of ACS patients potentiated platelet activation, an effect suppressed by the specific MMP-2-inhibitor, tissue inhibitor of MMP-2 (TIMP-2). CONCLUSION Matrix metalloproteinase-2 is released in the coronary circulation of ACS patients, derives in part from activated platelets, and may contribute to sustained intracoronary platelet activation.

Dominant inheritance of a novel integrin β3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families

Glanzmann’s thrombasthenia is a bleeding disorder caused by mutations of the ITGA2B or ITGB3 genes. This paper describes two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel mutation in the ITGB3 gene, encoding the integrin β chain β3. This report adds to previous studies of mutant alleles which illustrate the importance of signaling via this integrin for platelet function as well as for platelet formation. Background Defects of integrin αIIbβ3 are typical of Glanzmann’s thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann’s thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease. Design and Methods We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin β3-gene (ITGB3) mutation. Results The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface αIb β3, impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated αIIbβ3 upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective αIIbβ3-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647–686 of the βTD ectodomain of integrin β3. Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome. Conclusions This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin β3, and its βTD domain, in platelet formation and function.

Patients with primary antiphospholipid antibody syndrome and without associated vascular risk factors present a normal endothelial function.

INTRODUCTION Primary antiphospholipid antibody syndrome (PAPS) is characterized by venous or arterial thrombosis and positive antiphospholipid antibodies. It is controversial whether PAPS patients have early atherosclerosis. Endothelial dysfunction is an early event in the natural history of atherosclerosis. Aim of our study was to compare endothelial function of patients with PAPS and no associated risk factors with that of age- and sex-matched controls. MATERIALS AND METHODS Patients with PAPS, carefully selected to exclude all known risk factors for cardiovascular diseases, estrogen therapy, pregnancy, intake of drugs affecting endothelial function, vitamins or antioxidants, were included in a case-control study. Controls were age- (+/-5 years) and sex-matched subjects with the same exclusion criteria but without PAPS. Flow-mediated dilation of the brachial artery and some plasmatic markers of endothelial and platelet activation were measured. Measures are expressed as mean+/-SEM. RESULTS Twenty cases (mean age 42+/-4.0 years, 11 females) and 39 controls (mean age 41+/-2.9, 22 females) were studied. FMD was 5.7+/-0.8% in cases (95% CI: 4.1 to 7.3) and 6.8+/-0.5% (5.7 to 7.9) in controls (p=NS). Plasma von Willebrand factor was 128+/-11.3% and 134.2+/-16.1% in cases and controls, respectively (p=NS). Soluble P-selectin and soluble CD40L were 94.1+/-4.9 ng/ml and 0.7+/-0.1 ng/ml in cases and 87.7+/-4.0 ng/ml and 1.0+/-0.2 in controls, respectively (p=NS). In a substudy, circulating progenitor and mature endothelial cells were comparable between the two groups. CONCLUSIONS Endothelial function in patients with PAPS and no associated risk factors is similar to that of age- and sex- matched controls. These data suggest that the alterations leading to thrombosis in PAPS concern primarily the clotting system.

In vivo platelet activation and platelet hyperreactivity in abacavir-treated HIV-infected patients

Abacavir (ABC) has been associated with ischaemic cardiovascular events in HIV-infected patients, but the pathogenic mechanisms are unknown. Aim of our study was to assess whether ABC induces in vivo platelet activation and ex vivo platelet hyper-reactivity. In a retrospective, case-control study, in vivo platelet activation markers were measured in 69 HIV-infected patients, before starting therapy and after 6-12 months of either ABC (n=35) or tenofovir (TDF) (n=34), and compared with those from 20 untreated HIV-infected patients. A subgroup of patients was restudied after 28-34 months for ex vivo platelet reactivity. In vivo platelet activation markers were assessed by ELISA or flow cytometry, ex vivo platelet reactivity by light transmission aggregometry (LTA) and PFA-100®. Thein vitro effects of the ABC metabolite, carbovir triphosphate, on aggregation and intra-platelet cGMP were also studied. sPLA2, sPsel and sGPV increased significantly 6-12 months after the beginning of ABC, but not of TDF or of no treatment. Ex vivo platelet function studies showed enhanced LTA, shorter PFA-100® C/ADP closure time and enhanced platelet expression of P-sel and CD40L in the ABC group. The intake of ABC blunted the increase of intraplatelet cGMP induced by nitric oxide (NO) and acutely enhanced collagen-induced aggregation. Preincubation of control platelets with carbovir triphosphate in vitro enhanced platelet aggregation and blunted NO-induced cGMP elevation. In conclusion, treatment with ABC enhances in vivo platelet activation and induces platelet hyperreactivity by blunting the inhibitory effects of NO on platelets. These effects may lead to an increase of ischaemic cardiovascular events.

Hyperglycemia-Induced Platelet Activation in Type 2 Diabetes Is Resistant to Aspirin but Not to a Nitric Oxide–Donating Agent

OBJECTIVE Acute, short-term hyperglycemia enhances high shear stress–induced platelet activation in type 2 diabetes. Several observations suggest that platelets in type 2 diabetes are resistant to inhibition by aspirin. Our aim was to assess comparatively the effect of aspirin, a nitric oxide–donating agent (NCX 4016), their combination, or placebo on platelet activation induced by acute hyperglycemia in type 2 diabetes. RESEARCH DESIGN AND METHODS In a double-blind, placebo-controlled, randomized trial, 40 type 2 diabetic patients were allocated to 100 mg aspirin once daily, 800 mg NCX 4016 b.i.d., both of them, or placebo for 15 days. On day 15, 1 h after the morning dose, a 4-h hyperglycemic clamp (plasma glucose 13.9 mmol/l) was performed, and blood samples were collected before and immediately after it for platelet activation and cyclooxygenase-1 (COX-1) inhibition studies. RESULTS Acute hyperglycemia enhanced shear stress–induced platelet activation in placebo-treated patients (basal closure time 63 ± 7.1 s, after hyperglycemia 49.5 ± 1.4 s, −13.5 ± 6.3 s, P < 0.048). Pretreatment with aspirin, despite full inhibition of platelet COX-1, did not prevent it (−12.7 ± 6.9 s, NS vs. placebo). On the contrary, pretreatment with the NO donor NCX 4016, alone or in combination with aspirin, suppressed platelet activation induced by acute hyperglycemia (NCX 4016 +10.5 ± 8.3 s; NCX 4016 plus aspirin: +12.0 ± 10.7 s, P < 0.05 vs. placebo for both). Other parameters of shear stress–dependent platelet activation were also more inhibited by NCX 4016 than by aspirin, despite lesser inhibition of COX-1. CONCLUSIONS Acute hyperglycemia-induced enhancement of platelet activation is resistant to aspirin; a NO-donating agent suppresses it. Therapeutic approaches aiming at a wider platelet inhibitory action than that exerted by aspirin may prove useful in patients with type 2 diabetes.