Diabetes and acute coronary syndromes

Diabetic patients with acute coronary syndromes (ACSs) are at a high risk for subsequent cardiovascular events but derive, at the same time, greater benefit from evidence-based therapy than non-diabetic individuals. State-of-the-art anti-thrombotic therapy includes a triple anti-platelet combination - aspirin, clopidogrel and glycoprotein (GP) IIb/IIIa receptor inhibitors - and unfractionated heparin or enoxaparin. For low- or medium-risk individuals, a treatment based on aspirin, clopidogrel and bivalirudin is a valuable alternative. Prasugrel, a new and more potent inhibitor of the platelet P2Y(12) receptor, has to be regarded as the most promising anti-thrombotic agent for diabetic patients with ACS. This agent may replace clopidogrel - and possibly GP IIb/IIIa inhibitors - in the future. In addition to aggressive anti-thrombotic therapy, diabetic patients should undergo systematic early invasive angiography if presenting with non-ST-segment elevation ACS, and immediate percutaneous coronary intervention if presenting with ST-segment elevation myocardial infarction. Indeed, the benefit derived from these strategies appears to be more pronounced in the diabetic population than in non-diabetic individuals. Despite the benefit, multiple surveys have demonstrated that, in the setting of ACS, diabetic patients receive evidence-based therapy less frequently than non-diabetic counterparts.

Stent thrombosis with ticagrelor versus clopidogrel in patients with acute coronary syndromes: an analysis from the prospective, randomized PLATO trial

BACKGROUND We aimed to describe the effects of ticagrelor versus clopidogrel on stent thrombosis in the Platelet Inhibition and Patient Outcomes (PLATO) trial. METHODS AND RESULTS Of 18 624 patients hospitalized for acute coronary syndromes, 11 289 (61%) had at least 1 intracoronary stent. Ticagrelor reduced stent thrombosis compared with clopidogrel across all definitions: definite, 1.37% (n=71) versus 1.93% (n=105; hazard ratio [HR], 0.67; 95% confidence interval [CI], 0.50-0.90; P=0.0091); definite or probable, 2.21% (n=118) versus 2.87% (n=157; HR, 0.75; 95% CI, 0.59-0.95; P=0.017); and definite, probable, and possible, 2.94% (n=154) versus 3.77 (n=201; HR, 0.77; 95% CI, 0.62-0.95). The reduction in definite stent thrombosis was consistent regardless of acute coronary syndrome type, presence of diabetes mellitus, stent type (drug-eluting or bare metal stent), CYP2C19 genetic status, loading dose of aspirin, dose of clopidogrel before randomization, and use of glycoprotein IIb/IIIa inhibitors at randomization. The reduction in stent thrombosis with ticagrelor was numerically greater for late (>30 days; HR, 0.48; 95% CI, 0.24-0.96) and subacute (4 hours-30 days; HR, 0.60; 95% CI, 0.39-0.93) compared with acute (<24 hours; HR, 0.94; 95% CI, 0.43-2.05) stent thrombosis or for patients compliant to therapy (ie, taking blinded study treatment ≥80% of the time) compared with less compliant patients. Randomization to ticagrelor was a strong independent inverse predictor of definite stent thrombosis (HR, 0.65; 95% CI, 0.48-0.88). CONCLUSION Ticagrelor compared with clopidogrel reduces the incidence of stent thrombosis in patients with acute coronary syndromes, with consistent benefit across a broad range of patient, stent, and treatment characteristics.

Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis

Background: A clinically relevant bleeding diathesis is a frequent diagnostic challenge, which sometimes remains unexplained despite extensive investigations. The aim of our work was to evaluate the diagnostic utility of functional platelet testing by flow cytometry in this context. Methods: In case of negative results after standard laboratory work-up, flow cytometric analysis (FCA) of platelet function was done. We performed analysis of surface glycoproteins (GP) Ibα, IIb, IIIa; P-selectin expression and PAC-1 binding after graded doses of ADP, collagen and thrombin; content/secretion of dense granules; ability to generate procoagulant platelets. Results: Out of 437 patients investigated with standard tests between January 2007 and December 2011, we identified 67 (15.3%) with high bleeding scores and non-diagnostic standard laboratory work-up including platelet aggregation studies. Among these patients FCA revealed some potentially causative platelet defects: decreased dense-granule content/secretion (n=13); decreased alpha-granule secretion induced by ADP (n=10), convulxin (n=4) or thrombin (n=3); decreased fibrinogen-receptor activation induced by ADP (n=11), convulxin (n=11) or thrombin (n=8); decreased generation of COAT-platelets, i.e. highly procoagulant platelets induced by simultaneous activation with collagen and thrombin (n=16). Conclusion: Our work confirms that storage pool defects are frequent in patients with a bleeding diathesis and normal coagulation and platelet aggregations studies. Additionally, flow cytometric analysis is able to identify discrete platelet activation defects. In particular, we show for the first time that a relevant proportion of these patients has an isolated impaired ability to generate COAT-platelets - a conceptually new defect in platelet procoagulant activity, that is missed by conventional laboratory work-up. © 2014 Clinical Cytometry Society.

Echicetin coated polystyrene beads: a novel tool to investigate GPIb-specific platelet activation and aggregation.

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.

Identification of platelet function defects by multi-parameter assessment of thrombus formation.

Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Normal platelet activation profile in patients with peripheral arterial disease on aspirin.

BACKGROUND Peripheral arterial disease (PAD) is a progressive vascular disease associated with a high risk of cardiovascular morbidity and death. Antithrombotic prevention is usually applied by prescribing the antiplatelet agent aspirin. However, in patients with PAD aspirin fails to provide protection against myocardial infarction and death, only reducing the risk of ischemic stroke. Platelets may play a role in disease development, but this has not been tested by proper mechanistic studies. In the present study, we performed a systematic evaluation of platelet reactivity in whole blood from patients with PAD using two high-throughput assays, i.e. multi-agonist testing of platelet activation by flow cytometry and multi-parameter testing of thrombus formation on spotted microarrays. METHODS Blood was obtained from 40 patients (38 on aspirin) with PAD in majority class IIa/IIb and from 40 age-matched control subjects. Whole-blood flow cytometry and multiparameter thrombus formation under high-shear flow conditions were determined using recently developed and validated assays. RESULTS Flow cytometry of whole blood samples from aspirin-treated patients demonstrated unchanged high platelet responsiveness towards ADP, slightly elevated responsiveness after glycoprotein VI stimulation, and decreased responsiveness after PAR1 thrombin receptor stimulation, compared to the control subjects. Most parameters of thrombus formation under flow were similarly high for the patient and control groups. However, in vitro aspirin treatment caused a marked reduction in thrombus formation, especially on collagen surfaces. When compared per subject, markers of ADP- and collagen-induced integrin activation (flow cytometry) strongly correlated with parameters of collagen-dependent thrombus formation under flow, indicative of a common, subject-dependent regulation of both processes. CONCLUSION Despite of the use of aspirin, most platelet activation properties were in the normal range in whole-blood from class II PAD patients. These data underline the need for more effective antithrombotic pharmacoprotection in PAD.

Sodium-Dependent Phosphate Transporters in Osteoclast Differentiation and Function

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

When Does Neoadjuvant Chemotherapy Really Avoid Radiotherapy? Clinical Predictors of Adjuvant Radiotherapy in Cervical Cancer.

BACKGROUND The aim of this study was to identify clinical variables that may predict the need for adjuvant radiotherapy after neoadjuvant chemotherapy (NACT) and radical surgery in locally advanced cervical cancer patients. METHODS A retrospective series of cervical cancer patients with International Federation of Gynecology and Obstetrics (FIGO) stages IB2-IIB treated with NACT followed by radical surgery was analyzed. Clinical predictors of persistence of intermediate- and/or high-risk factors at final pathological analysis were investigated. Statistical analysis was performed using univariate and multivariate analysis and using a model based on artificial intelligence known as artificial neuronal network (ANN) analysis. RESULTS Overall, 101 patients were available for the analyses. Fifty-two (51 %) patients were considered at high risk secondary to parametrial, resection margin and/or lymph node involvement. When disease was confined to the cervix, four (4 %) patients were considered at intermediate risk. At univariate analysis, FIGO grade 3, stage IIB disease at diagnosis and the presence of enlarged nodes before NACT predicted the presence of intermediate- and/or high-risk factors at final pathological analysis. At multivariate analysis, only FIGO grade 3 and tumor diameter maintained statistical significance. The specificity of ANN models in evaluating predictive variables was slightly superior to conventional multivariable models. CONCLUSIONS FIGO grade, stage, tumor diameter, and histology are associated with persistence of pathological intermediate- and/or high-risk factors after NACT and radical surgery. This information is useful in counseling patients at the time of treatment planning with regard to the probability of being subjected to pelvic radiotherapy after completion of the initially planned treatment.

How often parametrial involvement leads to post-operative adjuvant treatment in locally advanced cervical cancer after neoadjuvant chemotherapy and type C radical hysterectomy?

OBJECTIVE Parametrial involvement (PMI) is one of the most important factors influencing prognosis in locally advanced stage cervical cancer (LACC) patients. We aimed to evaluate PMI rate among LACC patients undergoing neoadjuvant chemotherapy (NACT), thus evaluating the utility of parametrectomy in tailor adjuvant treatments. METHODS Retrospective evaluation of consecutive 275 patients affected by LACC (IB2-IIB), undergoing NACT followed by type C/class III radical hysterectomy. Basic descriptive statistics, univariate and multivariate analyses were applied in order to identify factors predicting PMI. Survival outcomes were assessed using Kaplan-Meier and Cox models. RESULTS PMI was detected in 37 (13%) patients: it was associated with vaginal involvement, lymph node positivity and both in 10 (4%), 5 (2%) and 12 (4%) patients, respectively; while PMI alone was observed in only 10 (4%) patients. Among this latter group, adjuvant treatment was delivered in 3 (1%) patients on the basis of pure PMI; while the remaining patients had other characteristics driving adjuvant treatment. Considering factors predicting PMI we observed that only suboptimal pathological responses (OR: 1.11; 95% CI: 1.01, 1.22) and vaginal involvement (OR: 1.29 (95%) CI: 1.17, 1.44) were independently associated with PMI. PMI did not correlate with survival (HR: 2.0; 95% CI: 0.82, 4.89); while clinical response to NACT (HR: 3.35; 95% CI: 1.59, 7.04), vaginal involvement (HR: 2.38; 95% CI: 1.12, 5.02) and lymph nodes positivity (HR: 3.47; 95% CI: 1.62, 7.41), independently correlated with worse survival outcomes. CONCLUSIONS Our data suggest that PMI had a limited role on the choice to administer adjuvant treatment, thus supporting the potential embrace of less radical surgery in LACC patients undergoing NACT. Further prospective studies are warranted.

Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.