Béla Nagy

The emerging value of P-selectin as a disease marker

Abstract Activated platelets are key components in many arterial disorders. P-selectin is an activation-dependent platelet receptor, which is also identified in endothelial cells. Together with E-and L-selectin it constitutes the selectin family. These transmembrane proteins have continued to attract great interest as they support rapid and reversible cell adhesion in flow systems and thus play an essential role in multicellular interactions during thrombosis and inflammation. Similarly to other lectins, selectins bind to different glycoconjugates with varying affinities. Protein ligands, equipped with the appropriate carbohydrate and sulfate moieties for P-selectin binding, have been identified in normal peripheral blood leukocytes and several non-hematopoietic organs, as well as on cancer cells. For diagnostic purposes, P-selectin can readily be detected on the platelet surface by flow cytometry and by ELISA as a soluble ligand in the plasma. Along with other markers, these data can be used in the assessment of platelet activation status. Such results bear clinical significance since P-selectin has been implicated in the pathogenesis of widespread disorders including coronary artery disease, stroke, diabetes and malignancy.

Impaired activation of platelets lacking protein kinase C-θ isoform

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-theta is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC-theta-selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PAR-induced aggregation, dense and alpha-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-theta, platelet aggregation and secretion were also impaired. PKC-mediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-theta RACK peptide, which may contribute to the lower levels of granule secretion when PKC-theta function is lost. Furthermore, the level of JON/A binding to activated alpha(IIb)beta(3) receptor was also significantly decreased in PKC-theta(-/-) mice compared with wild-type littermates. PKC-theta(-/-) murine platelets showed significantly lower agonist-induced thromboxane A(2) (TXA(2)) release through reduced extracellular signal-regulated kinase phosphorylation. Finally, PKC-theta(-/-) mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl(3) in vivo thrombosis model compared with wild-type mice. In conclusion, PKC-theta isoform plays a significant role in platelet functional responses downstream of PAR and GPVI receptors.

Protein Kinase Cδ Differentially Regulates Platelet Functional Responses

Objective—Protein Kinase C delta (PKC&dgr;) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKC&dgr; in platelets. Methods and Results—We evaluated the role of PKC&dgr; in platelets using two approaches—pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (&dgr; V1-1)TAT, and in the murine platelets lacking PKC&dgr;, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKC&dgr;. However, PKC&dgr; had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKC&dgr; null mouse platelets and in human platelets pretreated with (&dgr; V1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKC&dgr;−/− mice occluded similar to their wild-type littermates. Conclusions—Hence, we conclude that PKC&dgr; differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKC&dgr; deficiency does not affect the thrombus formation in vivo.

Elevated human epididymis protein 4 concentrations in chronic kidney disease

Background Human epididymis protein 4 (HE4) has recently become an available tumour biomarker for detecting ovarian cancer along with the standard cancer antigen 125 (CA125). However, it is unknown if the levels of HE4 and CA125 may be altered in subjects who have impaired renal function with no ovarian disorders. Methods In 113 female patients at different stages of chronic kidney disease (CKD) with no ovarian and lung cancer and 68 subjects with normal renal and ovarian function, HE4 and CA125 concentrations were analysed by using chemiluminescent microparticle immunoassay (Architect®, Abbott) and electrochemiluminescent immunoassay (Modular E170®, Roche), respectively. Renal function was evaluated by measuring serum creatinine and urea concentrations (Cobas Integra-800®, Roche). Estimated glomerular filtration rate (eGFR in mL/min/1.73 m2) was calculated by the 4v-MDRD formula. Results Significantly increased HE4 concentrations (P < 0.0001) were found in individuals with differently decreased eGFR values (<90 mL/min/1.73 m2) compared with clinical controls. CA125 serum concentration was higher than normal in subjects with CKD3 (eGFR = 30–59 mL/min/1.73 m2), but significant elevation (P = 0.006) in CA125 concentrations was seen only in those who had severe renal failure (CKD4-5; eGFR < 30 mL/min/1.73 m2). These tendencies were independent of age in our study cohort, and seemed to be more evident among women in premenopausal status. Conclusions HE4 concentrations may be elevated in CKD patients with no ovarian and lung cancer. Thus, HE4 results should be interpreted cautiously in women with renal disorders.

Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients

The role of platelets in the development of atherosclerosis and obesity-related prothrombotic state is still under investigation. In this cross-sectional cohort study, we measured the levels of different platelet activation markers and evaluated their relationship with carotid intima-media thickness (IMT) along with other atherosclerotic risk factors in obese patients with or without atherosclerotic co-morbidities. We enrolled 154 obese patients, including 98 with either hypertension, type 2 diabetes mellitus or dyslipidaemia, 56 without these co-morbidities and 62 age- and sex-matched healthy controls. Platelet P-selectin expression and the number of platelet-derived microparticles (PMPs) were measured by flow cytometry; soluble P-selectin levels were analysed by ELISA and Thr715Pro P-selectin polymorphism was determined by PCR-RFLP. Carotid IMT was examined by ultrasonography. The levels of platelet activation parameters were significantly elevated in all obese subjects with increased carotid IMT compared to healthy controls. There was no effect of Thr715Pro genotype on soluble P-selectin levels in obese individuals contrary to normal subjects. Significant and positive association was revealed between carotid IMT and platelet P-selectin (p<0.0001), soluble P-selectin (p=0.039) and PMP (p=0.0001) levels. After adjusting for multiple variables, independent association was found between soluble P-selectin and fibrinogen (p=0.007), PMP levels and body mass index (p<0.0001) as well as platelet P-selectin and carotid IMT (p=0.012) plus plasminogen activator inhibitor-1 (p=0.009). In conclusion, P-selectin and PMP levels showed positive associations with abnormal carotid IMT and other risk factors in obesity suggesting a critical role of enhanced platelet reactivity in atherosclerotic wall alteration.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a Gq blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y1 and Gq knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin αIIbβ3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a αIIbβ3 receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, αIIbβ3 failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y1 receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by αIIbβ3 integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

Chemotactic and random movement of human newborn monocytes

Boydens method was used to assess the chemotactic and random mobility of monocytes from healthy newborns, children aged 3–8 years, and adults aged 20–28. Monocyte chemotaxis in the newborn was decreased in comparison with that of control groups. No difference was observed in random cell mobility in the groups examined. Monocyte chemotactic activity in infants over 3 years of age was the same as in adults. According to our results, the leading-front and lower-surface evaluation methods, in newborns at least, do not give the same information on chemotaxis.

Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia.

Summary.u2002 Background:u2002In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective:u2002This study was carried out to investigate the contribution of the ADP receptor P2Y12‐mediated pathway to platelet hyperreactivity due to hypercholesterolemia. Methods:u2002Low‐density lipoprotein receptor‐deficient mice and C57Bl/6 wild‐type mice were fed on normal chow and high‐fat (Western or Paigen) diets for 8u2003weeks to generate differently elevated cholesterol levels. P2Y12 receptor‐induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation and secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal‐regulated protein kinase (ERK) and Akt were analyzed. Results:u2002Plasma cholesterol levels ranged from 69u2003±u200310 to 1011u2003±u2003185u2003mgu2003dL−1 depending on diet in mice with different genotypes. Agonist‐dependent aggregation, dense and α‐granule secretion and JON/A binding were gradually and significantly (Pu2003<u20030.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels, even if elevated thromboxane generation was blocked. These functional responses were induced via increased levels of Gi‐mediated ERK and Akt phosphorylation in hypercholesterolemic mice vs. normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR‐C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared with normocholesterolemic controls. Conclusions:u2002These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia.

Binding of plasma factor XIII to thrombin-receptor activated human platelets

Platelet-bound coagulation factor XIII (FXIII) is targeted and concentrated at the site where platelet-rich thrombi are formed. Previous studies were in disagreement about the nature of FXIII binding to platelets. In this study, thrombin-receptor activating peptide (TRAP)-stimulated human whole blood and washed platelets were analysed by flow cytometry for the binding of FXIII using a monoclonal antibody against the A subunit of FXIII (FXIII-A). Here, we demonstrate that FXIII-A positivity significantly increased on activated platelets in whole blood compared to unstimulated sample, but not in washed platelets. GPIIb/IIIa receptor plays an essential role in FXIII binding, as fibrinogen receptor antagonist eptifibatide and fibrinogen binding inhibitor RGDS tetrapeptide significantly prevented the binding of FXIII. Furthermore, stimulated platelets from a patient with severe type I Glanzmann thrombasthenia showed insignificant FXIII-A positivity versus healthy controls. In addition, basal negligible amount of FXIII on washed platelets was only slightly increased when highly purified plasma FXIII (FXIII-A(2)B(2)) was added upon platelet activation by TRAP. Similarly, no remarkable FXIII-A positivity was observed when we used FXIII-A(2)B(2) with gammaA/gammaA fibrinogen. However, gammaA/gamma fibrinogen significantly augmented FXIII binding on TRAP-stimulated platelets in the presence of non-activated FXIII-A(2)B(2). We conclude that FXIII-A(2)B(2) of plasma origin binds to thrombin-receptor activated platelets via GPIIb/IIIa receptor-bound fibrinogen with gamma-chain and is not capable of direct platelet binding.

Serum human epididymis protein 4 (HE4) as a tumor marker in men with lung cancer

Abstract Background: Human epididymis protein 4 (HE4) is a reliable tumor marker for ovarian cancer, but only limited data are available on HE4 levels in lung malignancies. Methods: HE4 levels were measured at diagnosis in 98 men with lung cancer at different stages of the disease, and these results were compared to an age-matched healthy male cohort (n=98). The concentrations of classical tumor markers were also determined, and their efficacy was compared to that of HE4. Results: Compared to healthy controls, patients with lung neoplasm showed significantly higher HE4 levels [118.2 (80.6–150.1) pmol/L vs. 62.2 (47.2–76.1) pmol/L; p<0.001]. Although age and smoking modulated HE4 levels in the healthy cohort, no such effect was observed in the patient population. The area under the receiver operating characteristic curve (ROC-AUC) for HE4 was 0.848 (95% CI 0.792–0.904) for differentiating lung cancer patients from healthy controls, with a cut-off value of 97.6 pmol/L (sensitivity: 64.3%, specificity: 95.9%). HE4 levels were significantly elevated in all stages of lung cancer, and even in patients without clinical symptoms (p<0.05), but no difference was found between the different histological subgroups. A significant correlation was found between HE4 values and the tumor size determined by CT/MRI (Spearman’s ρ=0.227, p=0.030). The combination of HE4 with CEA and CA 125 considerably enhanced the diagnostic efficacy [ROC-AUC: 0.963 (95% CI 0.937–0.990), sensitivity: 91.8%, specificity: 92.8%]. Conclusions: Our data suggest that serum HE4, especially in combination with CEA and CA 125, qualifies as a surrogate diagnostic marker in men with lung cancer.