Influence of aflibercept on platelet activation profile.

Aflibercept appears to accumulate in systemic circulation following intravitreal injections in therapy of neovascular age-related macular degeneration. This gives raise to the question of whether aflibercept affects platelets and their function such as activation and aggregation, which are substantial in the pathogenesis of an arterial thromboembolic event (ATE). In order to determine the effect of aflibercept in platelet activation, platelets from healthy volunteers were treated with aflibercept and its solvents at equal concentrations (0.04 µg/mL - 4 µg/mL - 40 µg/mL - 400 µg/mL - 4 mg/mL) for 10 and 30 min before addition of agonists. IgG1 antibody was used as a control. The surface expression of GPIIb/IIIa, P-selectin, and platelet-bound stromal-cell-derived factor-1, which are potential blood biomarkers for ATEs, was determined on resting and activated platelets by the multispectral imaging flow cytometry, combining the features of flow cytometry with fluorescence microscopy. Platelet aggregation was assessed with light transmission aggregometry. To determine whether aflibercept directly interacts with platelets, aflibercept was labeled with the fluorescence FITC. Co-treatment of platelets with thrombin or PAR-4-AP and aflibercept resulted in increased activation of the fibrinogen receptor GPIIb/IIIa in comparison to controls (P < 0.05). Interestingly, the expression of platelet-derived P-selectin and SDF-1 was not affected by aflibercept, except thrombin-activated CD62P with 0.04 µg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.54, IC = 1.201-1.879 vs. MSI = 1.37, IC = 1.136-1.604 [P = 0.031]) and SDF-1 with 4 mg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.971, IC = 1.206-2.737 vs. MSI = 1.200, IC = 0.738-1.662 [P = 0.041]). Although the levels of platelet-bound aflibercept-FITC were significantly increased in all activated platelets, no effect was observed in platelet aggregation. Albeit no impact of aflibercept was found on platelet aggregation under the studied experimental conditions, the increased activation of the fibrinogen receptor GPIIb/IIIa and the presence of a direct interaction between aflibercept and platelets may partially explain the risk of ATE in patients under aflibercept treatment due to FcγRIIa mediated αIIbß3 outside-in integrin signaling and transport of aflibercept into platelets. Therefore, the Fc domain seems to be involved in interactions between aflibercept and platelets. Further research is needed to explain the role of Fc containing aflibercept in the pathogenesis of drug-associated vascular events involving platelets, coagulation cascade, extracellular matrix proteins and other cells.

Combination of high on-treatment platelet aggregation and low deaggregation better predicts long-term cardiovascular events in PCI patients under dual antiplatelet therapy.

High on-treatment platelet reactivity is associated with short-term major cardiovascular (CV) events in patients undergoing percutaneous coronary intervention (PCI). Maximum and final aggregation assessed by light transmission aggregometry (LTA) have both been used to predict short-term outcome after PCI, however their long-term prognostic impact remains controversial. There is currently no information regarding the prognostic role of deaggregation and its added value in combination with established aggregation parameters. About 1279 patients with symptomatic coronary artery disease (CAD) undergoing PCI were enrolled in this monocentric study. On-treatment platelet aggregation under clopidogrel maintenance therapy, as well as deaggregation was determined by maximum and final aggregation (5 min after adding of the agonist). Deaggregation was defined as slope of the tangent between Aggmax +0.5 min. Primary endpoints were the composite of myocardial infarction, stroke, and CV death or stent thrombosis according to the ARC criteria. Low deaggregation, defined as values in the lowest tertile (<1.5), was more frequent in patients with acute coronary syndromes (ACS) compared to patients with stable angina pectoris (SAP), ACS: 29.6% vs. SAP: 22.0%, p = 0.001. The combination of high on-treatment platelet reactivity, defined by the upper tertile of Aggmax and low deaggregation, was associated with significantly increased risk for combined long-term CV events. The combination of low deaggregation and high on-treatment platelet reactivity is associated with higher risk for recurrent events in patients with CAD undergoing PCI. Thus, deaggregation might be a more sensitive parameter providing added value in terms of risk prediction for long-term recurrent CV events in relation with established aggregation parameters.

Expression of stromal-cell-derived factor-1 (SDF-1): a predictor of ischaemic stroke?

BACKGROUND AND PURPOSE: Platelet stromal-cell-derived factor-1 (SDF-1) plays a pivotal role in angiogenesis and the regeneration of ischaemic tissue through the regulation of haematopoietic progenitor cells and is upregulated at the sites of vascular injury and platelet activation. Thus, SDF-1 has recently been discussed as a predictor in ischaemic diseases such as acute myocardial infarction. However, no clinical data pertinent to the investigation of the platelet SDF-1 expression in patients with stroke are available. METHODS: We consecutively evaluated 196 patients who were admitted to the stroke unit with symptoms suspected for stroke. Surface expression of the platelet activation markers (P-selectin and GPIb) and the expression of platelet-bound SDF-1 were determined by two-colour whole blood flow cytometry. RESULTS: Patients with transient ischaemic attack (TIA) as well as with ischaemic stroke showed similar levels of SDF-1 expression on hospital admission compared with patients with non-ischaemic (NI) events and with 30 healthy controls (TIA (mean fluorescence intensity±SD): 31.5±18.2 vs. NI: 26.4±15.7; P=0.361; stroke: 28.7±19.8 vs. NI; P=0.943; control: 26.1±11.3; P>0.05 compared with all). Platelet SDF-1 expression showed a trend with the severity of stroke according to National Institute of Health Stroke Scale score (r=0.125; P=0.085), but significantly correlated with the peak levels of C-reactive protein (r=0.218; P=0.002) and with the levels of platelet activation (P-selectin: r=0.389; P=0.001). Multifactorial analysis of covariance revealed a significant influence on platelet SDF-1 expression by smoking (P=0.019). CONCLUSIONS: Platelet SDF-1 surface expression did not show any significant difference in patients with TIA and ischaemic stroke compared with patients with NI events. Thus, single biomarker evaluation of platelet SDF-1 surface expression is not helpful to predict ischaemic stroke.

Expression of platelet glycoprotein VI is associated with transient ischemic attack and stroke.

BACKGROUND AND PURPOSE: Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation. We aimed to investigate GPVI in patients presenting with symptoms of acute cerebrovascular disease and to define GPVI as biomarker for acute stroke. METHODS: We consecutively evaluated 205 patients, who admitted the stroke unit with symptoms for stroke. Surface expression of the platelet activation markers (GPVI, CD62P, GPIb) was determined by two-color whole blood flow cytometry. RESULTS: Patients with transient ischemic attack (TIA) (n = 18; 8.8%) as well as with stroke (n = 133; 64.9%) showed a significantly enhanced GPVI expression (mean fluorescence intensity +/- SD) on admission compared to patients with non-ischemic (NI) events (n = 54; 26.3%) (TIA: 20.9 +/- 7.1 vs. NI: 16.2 +/- 3.9; P = 0.002; stroke: 20.4 +/- 5.7 vs. NI; P = 0.002). Neither CD62P nor GPIb surface expression showed a significant difference. Logistic regression analysis revealed that on admission GPVI was associated with stroke independent of conventional laboratory markers such as C-reactive protein, blood glucose, and creatine kinase. Using a receiver operating characteristic curve on GPVI, we have determined the cut off value of 18.2 for stroke. Thus, patients with enhanced GPVI expression levels (>or=18.2) had a 2.4-fold relative risk for stroke. Patients with elevated platelet GPVI expression level had a poorer clinical outcome in cumulative event-free survival for stroke, myocardial infarction, and cerebro-/cardiovascular death at 3-month follow-up (log rank; P = 0.045). CONCLUSIONS: These findings indicate that platelet GPVI surface expression is significantly enhanced in patients with TIA and stroke compared to patients with NI events. Determination of platelet-specific GPVI may be useful as an early biomarker for cerebral ischemia.

Type I collagen biomarkers in the diagnosis of bone metastases in breast cancer, lung cancer, urinary bladder cancer and prostate cancer. Comparison to CEA, CA 15-3, PSA and bone scintigraphy.

PURPOSE: In this study we evaluated the clinical usefulness of serum pro-I collagen peptide (PICP) and I collagen telopeptide (ICTP) as indicators of early bone metastases in patients with breast (BC), lung (LC), urinary bladder (UBC) and prostate cancer (PC). PATIENTS AND METHODS: 305 patients were examined. 145 had histologically confirmed BC (92 with bone metastases), 20 UBC (6 with bone metastases), 11 LC (3 with bone metastases) and 129 PC (68 with bone metastases). In BC patients we compared the PICP and ICTP levels with those of CA 15-3, CEA and bone scintigraphy. Patients with LC and UBC had PICP and ICTP measurements, PC patients had serum PICP, prostate specific antigen (PSA) measurements and bone scans. 104 healthy individuals served as controls. RESULTS: ICTP and CA 15-3 levels were significantly higher in patients with BC and bone metastases in comparison to patients without metastases (p <0.05), while PICP and CEA were only marginally higher. Significant correlation was observed between existence of bone metastases and ICTP levels (p <0.05). The sensitivity of PICP, ICTP, CEA and CA 15-3 was 28.1, 48.6, 42, and 78%, respectively and specificity was 83.9, 94, 65 and 86%, respectively. ICTP and CA 15-3 were the most reliable markers for early diagnosis of bone metastases in BC. PICP alone or with ICTP were not sensitive enough. Only CA 15-3 showed sensitivity 78% and specificity 86%. When combined CA 15-3, ICTP and CEA the sensitivity and specificity increased to 82% and 96%, respectively. Furthermore, PICP and PSA levels were significantly higher in patients with PC and bone metastases in comparison to patients with benign prostate hyperplasia (BPH) (p <0.0001) or in patients with PC without bone metastases (p <0.0005 for PICP and p <0.0001 for PSA). The co-evaluation of PICP and PSA improved the sensitivity (78%), specificity (96%), accuracy (97%) and positive predictive value (97%). In LC patients, ICTP levels differed significantly between patients with and without bone metastases (p=0.025). In UBC patients, PICP levels differed significantly between patients with and without bone metastases (p=0.017). CONCLUSION: ICTP and CA 15-3 are the most reliable markers for early diagnosis of bone metastases in BC patients. PICP could be useful for diagnosing early bone metastases of PC and combined with PSA and bone scan can be an additional tool in the follow-up of PC patients. For LC patients, ICTP showed a significant difference in the discrimination of patients with and without bone metastases. In UBC patients, PICP showed a significant difference in the discrimination of patients with and without bone metastases.

The Residual Platelet Aggregation after Deployment of Intracoronary Stent (PREDICT) score.

BACKGROUND: Recent studies suggest a high interindividual variability of response to clopidogrel associated with adverse cardiovascular outcome. Different clinical factors are considered to influence a persistent residual platelet aggregation (RPA) despite conventional antiplatelet therapy. OBJECTIVES: To investigate clinical factors that affect RPA after 600-mg clopidogrel loading in a large unselected cohort of patients with symptomatic CAD. METHODS: The study population included a consecutive cohort of 1,092 patients treated with coronary stenting for stable angina and acute coronary syndromes (ACS). Residual platelet activity was assessed by ADP (20 micromol L(-1))-induced platelet aggregation >or= 6 h after LD. Eleven clinical factors were included in the primary analysis. RESULTS: In multivariate regression analysis increased RPA was significantly influenced by ACS, reduced LV-function, diabetes mellitus, renal failure (creatinine > 1.5 mg dL(-1)), and age > 65 years. In a factor-weighed model the risk for high RPA increased with higher score levels (OR for patients with a score of 1-3, 1.21, 95% CI 0.7-2.1; score 4-6, OR 2.0, 95% CI 1.17-3.5; P = 0.01; score 7-9, OR 3.3, 95% CI 1.8-6.0). During a 30-day follow-up the incidence of major adverse events was higher in patients with RPA in the upper tertile (4.8% vs. 2.5% in the 2nd and 1.5% in the 1st tertile; P < 0.05). CONCLUSIONS: The PREDICT score provides a good tool to estimate residual platelet activity after clopidogrel LD by easily available patient details. Additionally, we demonstrate its association with short-term outcome. Thus, patients with a high score may benefit from intensified antiplatelet therapy by improved platelet inhibition and risk reduction for thromboischemic events.

Association between bone scintigraphy and serum levels of procollagen (I) and PSA in the detection of bone disease in prostate cancer patients.

PURPOSE: To evaluate the clinical usefulness of serum procollagen I carboxyterminal propeptide (PICP) and prostate specific antigen (PSA) in relation to bone scan results in Greek patients with prostate cancer (PC). PATIENTS AND METHODS: 108 patients (mean age 58+/-4.3 years; range 42-81) with PC and 52 healthy blood donors as control group were examined for serum PICP and PSA levels. The diagnosis of PC was confirmed histologically. Bone metastases were diagnosed in 68 of the patients with the use of (99m)Tc-MDP bone scan, while 40 patients had no bone metastases. During the one year follow-up new PICP and PSA measurements were obtained along with a new bone scan for all groups studied. RESULTS: The levels of serum PICP and PSA were significantly higher in patients with PC and bone metastases in comparison to patients with no bone metastases. The sensitivity and specificity of the combination of PICP and PSA were 78% and 96%, respectively. CONCLUSION: PICP could be useful for diagnosing early bone metastases of prostate adenocarcinoma and in combination with PSA and bone scan can be an additional tool in the follow-up of patients with PC.

Prolactin as a predictor of endothelial dysfunction and arterial stiffness progression in menopause.

Postmenopausal women are at increased risk for progression of arteriosclerosis and hypertension. Recent cross-sectional evidence suggests that high normal circulating prolactin levels may accelerate vascular ageing in menopause. Postmenopausal women (n=201) were consecutively recruited from a Menopause Clinic and re-evaluated in at least one follow-up visit within the next 3 years. Baseline circulating prolactin levels were measured while both baseline and follow-up vascular and biochemical measurements were performed. Endothelial function was assessed by flow-mediated dilation (FMD), aortic stiffness by pulse-wave velocity (PWV) and arterial wave reflections by applanation tonometry. Baseline prolactin significantly correlated with lower FMD at follow-up (P=0.005). After multivariable adjustment for age, follow-up time, blood pressure (BP), body mass index, smoking and medication, this correlation remained significant (P=0.003). In addition, baseline circulating prolactin levels were independently associated with changes in mean BP (ß=0.131, P=0.021), peripheral diastolic BP (ß=0.169, P=0.004) and new-onset hypertension (OR=1.235, P=0.001). Owing to significant interaction between baseline prolactin and age for changes in PWV over time (P=0.036), a subgroup analysis based on median age was performed. This analysis revealed that in women younger than 55 years, prolactin was an independent predictor of changes in PWV over time (P=0.008). In conclusion, high normal circulating prolactin levels predict changes in haemodynamic indices and worsening endothelial function in healthy postmenopausal women. Particularly in young postmenopausal women, prolactin predicts accelerated arterial stiffening.

K statistic as a measure of quality control in cervicovaginal cytology.

Quality assessment schemes are widespread in most branches of pathology but are uncommon in the more subjective areas of histopathology and cytology. Researchers in many fields have become increasingly aware of the observer as an important source of measurement error. The validity of any method of reporting evidence of an abnormal process in cellular material is based on the degree of correlation with the actual disease process as it exists in the tissue and its reproducibility. Correlations can be tested in retrospective studies in which diagnoses based on cellular evidence are matched against the disease process present in biopsy specimens. Correlations can also be tested by examination of a set of unknown cellular preparations obtained in the presence of proven disease. While reproducibility is indirectly related to correlation, it is meant to imply satisfactory utilization of the method by other groups of cytotechnologists and cytopathologists. While cytopathology will continue to play an important role as a screening technique for the detection of cancer of the uterine cervix, its usefulness in the study of the early manifestations of the disease process is yet to be realized on a universal basis.

Expression of laminin, type IV collagen and fibronectin molecules is related to embryonal skin and epidermal appendage morphogenesis.

Basement membrane zones are specialized sheets--like arrangements of extracellular matrix proteins and glycosaminoglycans, and act as an interface between parenchymal cells and support tissue. They separate epithelium, endothelium, muscle cells and Schwann cells from adjacent connective tissue stroma, and also from a limiting membrane in the central nervous system. They are involved in several cellular and biological processes, including adhesion, migration and cellular differentiation. Basement membranes have five major components: collagen type IV, laminin, heparan sulfate, entactin, and fibronectin. In addition, there are numerous minor and poorly characterized protein and glycosaminoglycan components. The various components of the basement membranes of the skin (collagen type IV, proteoglycans--heparan sulfate, laminin, entactin and fibronectin) are products of the epithelial (epidermal) cells. We studied immunohistochemically the origin, the first appearance and distribution of the adhesive extracellular glycoprotein laminin and the fibrillar proteins of the extracellular matrix collagen type IV and fibronectin in the basement membranes of fetal human skin between 12 to 21 weeks of gestational age. Additionally, we studied the expression of vimentin in the extracellular matrix of the epithelial/mesenchyme junction of the skin. This study demonstrates clearly that the expression of the antigens laminin, collagen type IV and fibronectin starts in the germinative epithelial cells of the skin at the bulbs of the hair follicles (12th week for fibronectin and 19th week for laminin and collagen type IV), and migrating progressively involves the epithelial epidermal cells of the covering skin, as well as, the basement membrane at the dermal-epidermal junction in that region (between 20 to 21 weeks of gestational age).

Human decidual cells can express the Hodgkin's cell-associated antigen Ki-1 (CD30) in spontaneous abortions during the first trimester of gestation.

CD30 (Ki-1) antigen has been considered to be expressed on hematopoietic cells including the ones of the recently described anaplastic large cell lymphoma (ALCL), the Reed-Sternberg (RS) cells of Hodgkin's disease and the scattered large parafollicular cells in normal lymphoid tissues. Since then, several reports have been published describing CD30 expression in non-hematopoietic and malignant cells, such as cultivated human macrophages, human decidual cells, histiocytic neoplastic cells, mesothelioma cells, embryonal carcinoma, and seminoma cells. In the present study, we investigated the immunohistochemical expression of CD30 antigen in 15 paraffin-embedded placentas from fetuses after spontaneous abortion in the first trimester of gestation (8th, 10th, and 12th week, respectively) using the monoclonal antibody Ber-H2. All the pregnant patients had been given hormonal medication to support gestation. In addition, a panel of monoclonal antibodies for the identification of leukocytes (CD45/LCA), B-lymphocytes (CD20/L-26), and T-lymphocytes (CD45RO/UCHLI) was performed. Our findings were correlated with those found in 15 placentas obtained from 15 fetuses at the same time, after therapeutic or voluntary abortions. This study demonstrates that, 1) decidual endometrial stromal cells are able to express the CD30 (Ki-1) antigen, 2) the expression of CD30 in decidual cells is higher in cases of hormonal administration (to support gestation), than that found in normal gestation. In the former cases (hormonal support of gestation), a mild mononuclear infiltration of the decidua by UCHLI (T marker) positive cells, accompanies the CD30 positive cells.

Pleiotropic effects of the acute and chronic inhibition of the renin-angiotensin system in hypertensives.

Renin-angiotensin system (RAS) inhibition may exert beneficiary pleiotropic effects on heart hemodynamics in hypertensive patients. We aimed to assess these effects on coronary flow reserve (CFR) and left ventricular (LV) filling pressure after acute and long-term treatment. Thirty-nine patients (48.4±6.8 years) with newly diagnosed, never-treated essential arterial hypertension were consecutively recruited from an outpatient hypertension clinic. CFR in the left anterior descending artery and the ratio of mitral inflow E wave to the averaged mitral annulus tissue velocity of the E waves (E/e' ratio), as an estimate of LV filling pressure, were assessed by Doppler echocardiography. In the acute phase of the study, consecutive eligible patients were assigned to receive po Quinapril (Q) 20 mg (n=15) or Losartan (L) 100 mg (n=14) or no treatment (n=10) and were reexamined 2 h post treatment. In the chronic phase of the study, the patients were reevaluated after 1 month on the assigned treatment. During the acute phase, CFR (P=0.005) was significantly improved in the RAS inhibition as compared with the control group, independently of blood pressure (BP) changes. The E/e' ratio was also marginally improved (P=0.053), but this effect was more pronounced in patients with E/e' ratio>8 (P=0.005). CFR and E/e' ratio were also improved after 1 month of treatment, particularly in responders after the acute phase. In hypertensive patients, RAS inhibition acutely improved CFR and E/e' ratio independently of BP changes. An acute positive response in these parameters was closely related to sustained improvement after 1 month of single-drug treatment.

Platelets modulate atherogenesis and progression of atherosclerotic plaques via interaction with progenitor and dendritic cells.

Platelets not only play a role in the late complications of atherosclerosis, but are also essential in its initiation, interacting with endothelial cells and leukocytes. Platelet adhesion to injured or atherosclerotic vessels is critical for the initiation of atherosclerotic lesion formation in vivo. Increasing evidence has recently highlighted the role of progenitor cells in inflammation, atherogenesis, and atheroprogression. Recruitment of progenitor and dendritic cells to sites of vascular injury is poorly understood so far. Both human progenitor and dendritic cells significantly adhere to platelets, indicating that platelets adherent to collagen or to endothelial cells can serve as a bridging mechanism directing circulating progenitor and dendritic cells to sites of impaired vasculature. Moreover, platelets regulate differentiation of progenitor cells to endothelial cells or macrophages and foam cells and modulate essential functions of dendritic cells, including their activation, differentiation and apoptosis in vitro. This review describes recent findings on platelet interaction with progenitor cells or dendritic cells and discusses potential consequences of this interaction in atherosclerosis.