Platelet Extracellular Vesicles in Cord Blood of Term and Preterm Newborns Assayed by Flow Cytometry: the Effect of Delay in Sample Preparation and of Sample Freezing.

Plasma levels of circulating platelet extracellular vesicles (PEVs) are an emerging marker of platelet activation, thrombosis, inflammation, and endothelial dysfunction. Analysis of PEVs in cord blood of preterm newborns may reflect the underlying pathology and possibly serve as a new diagnostic and prognostic tool. However, collection, preparation and analysis of cord blood samples in clinical settings is a logistically complex process. We have studied the effect of delay in sample preparation and sample freezing on the PEV analysis by flow cytometry. PEVs in the cord blood plasma were identified after double labelling with monoclonal antibodies CD36+CD41 or CD41+CD62. Both, the delay and the freezing significantly affected the count and often also fluorescence of the detected PEVs. Additionally, our pilot study utilizing fresh cord blood samples of term and preterm newborns demonstrated significantly decreased CD36 and CD62 PEV fluorescence in preterm newborns. Our data highlight the importance of pre-analytical steps in the analysis of cord blood PEVs and suggest that not only the count, but also the level of PEV fluorescence may have possible diagnostic potential.

Production, purification and oxidative folding of the mouse recombinant prion protein.

The method leading to overexpression of the full-length mouse recombinant prion protein (mrPrP 23-231) in the cytoplasm of E. coli as a his-PrP fusion protein and its effective purification using affinity chromatography is described. A typical yield of the method was 8-10 mg his-mrPrP per L of the bacterial culture. The purity of purified protein was > 95 %. The purified his-mrPrP was converted to a soluble form and its folding to alpha-helical and beta-sheet conformations was studied. The properties of differently folded mrPrP were determined by measuring their circular dichroism spectra, partial resistance to cleavage by proteinase K and by centrifugation in sucrose gradient.

Expression of cellular prion protein on blood cells: potential functions in cell physiology and pathophysiology of transmissible spongiform encephalopathy diseases.

The cellular prion protein (PrPc) holds a central role in the pathophysiology of transmissible spongiform encephalopathies (TSE). The hallmark of these progressive neurodegenerative diseases is the accumulation of the protease-resistant, pathologic conformation of prion protein (PrPres) in the CNS. The conformational change is thought to be propagated by a template-like effect in which a normal prion protein (PrPc) interacts with its PrPres isoform and assumes the pathologic conformation. In its natural conformation, the prion protein is expressed on many different cell types, but its physiological function has yet to be clearly defined. PrPc expressed on blood cells or present in plasma may contribute to the transport of TSE infectivity found in blood of infected animal models. We examine the expression of PrPc on human and animal blood cells and its potential functional roles and discuss studies of transfusion-mediated transmission of TSE infectivity in animals.

Different levels of prion protein (PrPc) expression on hamster, mouse and human blood cells.

The host prion protein, PrPc, and its conformationally changed isoform, PrPsc, play an essential role in the transmissible spongiform encephalopathy (TSE) infections. The prion hypothesis postulates that PrPsc is the TSE infectious agent and that it serves as a template to convert host PrPc to additional PrPsc. Blood of experimentally TSE-infected rodents has been shown to contain TSE infectivity. If blood-borne TSE infectivity requires association with PrPc, differences in the distribution of PrPc in blood could affect the amount and distribution of blood-borne infectivity in different hosts. We have compared the distribution of PrPc on the peripheral blood cells of humans, hamsters and mice using quantitative flow cytometry. Human lymphocytes, monocytes and platelets displayed much greater quantities of PrPc than corresponding mouse cells. Mouse platelets did not express any detectable PrPc. A similar low level of PrPc was found on both human and mouse red blood cells. None of the hamster peripheral blood cells displayed detectable amounts of PrPc. If PrPc contributes to the propagation or transport of TSE infectivity in blood, the species differences in PrPc distribution reported here need to be considered when extrapolating the results of rodent TSE transmission studies with blood and blood components to humans.

Surface expression of major membrane glycoproteins on resting and TRAP-activated neonatal platelets.

Platelets of full-term newborns and those of healthy adult donors were compared for constitutive expression of surface glycoproteins (GP) Ia-IIa, GP Ib, GP IIb-IIIa, and GP IV and for their activation responses to an agonist by detection of surface expression of activation markers P-selectin and CD63. Resting neonatal platelets showed significantly lower expression of GP Ia-IIa, GP Ib, and GP IIb-IIIa. In contrast, the expression of GP IV was significantly higher compared with platelets of adults. The expression of activation markers P-selectin and CD63 was assessed after in vitro activating of platelets with 0-15 microM human thrombin receptor-activating peptide. At low concentrations of thrombin receptor-activating peptide, the extent of surface expression of activation markers did not differ significantly between adult and neonatal platelets. However, after activation with 15 microM thrombin receptor-activating peptide, the extent of surface expression of P-selectin and CD63 was significantly lower in neonatal platelets. Because of ethical reasons, our study was conducted on neonates with a moderate neonatal hyperbilirubinemia. The remote possibility that hyperbilirubinemia could influence the expression of platelet surface receptors and the reactivity of neonatal platelet cannot be excluded. The role of higher expression of GP IV on neonatal platelets, also seen in certain hematologic malignancies in adults, remains to be elucidated. The lower expression of platelet adhesive receptors and the limited ability to up-regulate granular glycoproteins may play a role in the impairment of function of neonatal platelets.

Increased expression of phosphatidylinositol-specific phospholipase C resistant prion proteins on the surface of activated platelets.

The surface expression of prion protein (PrP(C)) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two-fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrP(C) occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37 degrees C. In comparison, PrP(C) on the surface of platelets, activated at 22 degrees C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrP(C) and P-selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrP(C) was translocated from internal granules to the plasma membrane during activation, as is P-selectin. Platelet PrP(C) was not removed from the surface of platelets by phosphatidylinositol-specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrP(C).

Severe coagulopathy after a bite of a green bush viper (Atheris squamiger): case report and biochemical analysis of the venom.

A 34 year old male bitten by an adult Atheris squamiger snake developed symptoms of nausea, vomiting, diarrhea which were followed by drowsiness and impaired breathing. Local hemorrhage, edema and pain at the bite-site occurred, but no systemic bleeding or hemorrhagic diathesis developed. All clinical and laboratory parameters were in the normal range except for afibrinogenemia, thrombocytopenia and slight proteinuria. Replacement therapy (fibrinogen and platelet concentrates) and treatment of shock stabilized the patient within 2d and coagulation returned to normal. Atheris squamiger venom was subjected to biochemical and biological analysis. The LD50 of the venom was 5 mg/kg (mice, s.c.). It produced local hemorrhage corresponding to about 25% of the activity of puff adder venom (Bitis arietans). In vitro the venom had a fibrinogen-converting activity, it did not activate purified prothrombin but very likely contained a F V and Ca2+-dependent prothrombin activator. The venom exhibited strong platelet-aggregating activity, which was not inhibited by protease inhibitors and by EDTA or EGTA. The venom also aggregated acetylsalicylic acid treated platelets indicating, that the arachidonic acid pathway was not essential for activation. Rat serum rapidly inhibited the platelet-aggregating activity of the venom; human serum, however, had only a partial inhibitory effect. Preliminary experiments showed that platelet-aggregating activity may be separated from fibrinogen-converting activity by anion-exchange chromatography.

A comparative SAR study of thrombin receptor derived non peptide mimetics: importance of phenyl/guanidino proximity for activity.

Thrombin, the most potent physiological platelet agonist interacts with cells through a specific G protein-coupled receptor which has been cloned and sequenced. Synthetic thrombin receptor peptides (TRAPs) comprising the first 5 amino acids (SFLLR and SFLLR-NH2) of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity were found to cause full platelet aggregation. During the screening of novel thrombin receptor derived non-peptide mimetics in the platelet aggregation assay we found that 1-phenylacetyl-4-(6-guanidohexanoyl)-piperazine (1) and 1-(6-guanidohexanoyl)-4-(phenylacetylamidomethyl)-piperidine (2) exerted in vitro antagonist activities (56% and 40% correspondingly) as it is depicted by the platelet aggregation assay. Using Molecular Modeling, the synthetic compounds were overlayed with SFFLR. All three superimposed low energy structures had Phe and Arg amino acids in spatial close proximity. The superimposition results revealed that 1 resembled more the stereoelectronic environment of SFLLR than 2. This difference may be related to their different antagonist efficacy.

Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation.

Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600 s(-1) with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s(-1) almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to adsorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.

Photodynamic effects of meso-tetra (4-sulfonatophenyl)porphine on human leukemia cells HEL and HL60, human lymphocytes and bone marrow progenitor cells.

Meso-tetra(4-sulfonatophenyl)porphine (TPPS4), in combination with a light dose of 14 J cm-2, has a profound negative effect on the proliferation and viability of leukemia cells HL60 (human promyelocytic leukemia) and HEL (human erythroleukemia), the viability of normal lymphocytes and the colony-forming activity of human bone marrow progenitor cells. However, normal leukocytes (monocytes, granulocytes) are, to a large extent, resistant to photodynamic treatment (PDT). Whilst DNA fragmentation suggesting apoptosis is induced in HL60 cells, accumulation in the interphase of the cell cycle (G0/G1, G2/M) is mainly operative in the TPPS4-mediated PDT of HEL cells. The "dark" effect of TPPS4 on the cell viability is below 15% up to a concentration of 40 microM.

[The Bernard-Soulier syndrome. A hereditary functional and structural disorder of blood platelets]. / Bernarduv-Soulieruv syndrom. Hereditární funkcní a tvarove strukturální porucha krevních desticek.

BACKGROUND: The authors submit a clinical and laboratory description of a patient with Bernard-Soulier syndrome diagnosed in this country for the first time. The investigation comprises an analysis of haemostatic functions, selected structural indicators and the detailed morphology of thrombocytes. The analysis of defined inborn functional disorders of thrombocytes is a method for studying haemostatic mechanisms. METHODS AND RESULTS: In addition to standard haemostatic methods flow cytometry in used to assess the size of platelets, and using monoclonal antibodies against glycoproteins, their presence on the surface membrane is assessed. Thrombocytic glycoproteins are further analyzed using diagonal electrophoresis on sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE). The authors found medium thrombocytopaenia with the presence of giant thrombocytes which on elecronmicroscopic examination, with the occasional exception of endoplasmic membrane proliferation, do not display structural deviations. The assessed deviation of thrombocyte aggregation after ristocetin, suggesting a defect of the adhesive capacity of thrombocytes, is apparently due to reduction of the surface glycoprotein GPIb, proved by flow cytometry and SDS-PAGE. The drop of glycoprotein GPIb to 20% of normal values suggests a heterozygote type of disorder with a medium grade of haemorrhagic manifestations. CONCLUSIONS: This is the first case of Bernard-Soulier syndrome in the Czech literature. Its analysis provides evidence of the relationship of the adhesive function to the surface glycoprotein GPIb and confirm the effectiveness of the elaborated diagnostic methods.

Platelet membrane receptors during short cardiopulmonary bypass--a flow cytometric study.

To elucidate a mechanism of platelet dysfunction during extracorporeal circulation, we performed a study on the surface expression of platelet adhesive receptors (GPIb, GPIIb-IIIa) and activation markers (GMP140, GP53) during short cardiopulmonary bypass (CPB). Ten paediatric patients, age 6-13 years, with atrial or atrioventricular septal defects were studied. The mean CPB time was 52 min (21-110 min). During CPB, a significant drop in platelet count was observed, but not below 130 x 10(3)/microliter. The expression of platelet GPIb decreased slightly during CPB and the decrease was not significant. The decrease of GPIIb-IIIa was significant, but only in samples collected either at the end of CPB (89 +/- 13%, p < 0.05) or before leaving the operating room (74 +/- 14%, p < 0.05). The value of surface expression of platelet activation markers (GMP140, GP53) during CPB was in the range of values for resting platelets. Our results suggest that generalized CPB-induced defects of primary haemostasis are not directly connected to circulation of activated degranulated platelets or to loss of platelet adhesive receptors GPIb-IX and GPIIb-IIIa.

Characterization of platelet antigen for CD45RA monoclonal antibodies.

CD45RA monoclonal antibodies recognize the higher molecular weight isoforms (220 and 205 kDa) of leukocyte common antigen family (CD45), which are typically expressed on B cells and unstimulated T cells. We have found that there are at least three distinct CD45RA monoclonal antibodies which react with platelet 42 kDa (P42) intracellular protein antigen, which seems to be different from any to date described platelet proteins with similar molecular weight. This platelet antigen is a single chain protein, very likely not a glykoprotein, with isoelectrical point between 6.8 and 7.5. P42 does not seem to be a membrane protein and is not associated with platelet cytoskeleton. Results of immunofluorescence assay suggest that P42 may be redistributed to platelet surface after platelet aggregation.

[The fluorescence flow cytometry technique in the diagnosis of hereditary platelet disorders--a case of Glanzmann's thrombasthenia]. / Technika prutokové fluorescencní cytometrie v diagnostice dedicných destickových onemocnení--prípad Glanzmannovy trombastenie.

The authors describe a method for the diagnosis of heredital platelet membranopathies by means of monoclonal antibodies against the main membrane glycoproteins of thrombocytes, glycoprotein Ib and IIIa. The platelets are differentiated in the flow fluorocytometer from other blood cells by the typical optic profile caused by their size and granular character. Monoclonal antibodies are bound to the appropriate membrane glycoprotein and their amount is then detected by means of a secondary antibody labelled with fluorescein. The intensity of fluorescence of individual platelets is proportional to the number of molecules of the appropriate glycoprotein on their surface. By the above technique a case of Glanzmann's thrombasthenia was diagnosed, a rare hereditary haemorrhagic disease, characterized by the absence or abnormal function of glycoprotein complex IIb/IIIa the platelet receptor for fibrinogen.

Endothelial desquamating activity of rat synthetic fibrinopeptide B and its analogues "in vivo": identification of responsible sequence.

The endothelial desquamating activity of the synthetic rat fibrinopeptide B (ATTDSDKVDLSIAR-OH), and its analogues was studied "in vivo" after intravenous administration to rats. Rat fibrinopeptide B (FPB) caused a significant increase in the count of circulating endothelial cell carcasses at the dose of 100 nmol/kg. Maximal effect reaching about 270% of the normal value was achieved with the dose of 600 nmol/kg in 30 min. after the injection. No significant thrombocytopenia, no hemolysis and no other life-threatening complications were observed. The same endothelial desquamating effect was achieved with N-terminal FPB(1-7) peptide ATTDSDK-OH, but very low activity exhibited the two FPB mutant peptides: ATDSDKVDLSIAR-OH and ATTNSNK-OH. Our results indicate that N-terminal sequence (1-7) consisting of N-terminal "pig tail" (ATT), acid region (DSD) and basic amino acid (K) is responsible for endothelial desquamating activity of rat FPB. Similar corresponding sequences may be recognized in FPB of different species. The conservation of this common "active site" sequence is less apparent in primates.

Comparison of rat and human major platelet glycoproteins.

1. Using electrophoretic techniques combined with various detection methods we ascribed rat platelet glycoproteins (GPs) related to human GPIb, GPIIb and GPIIIa. 2. Rat GPIIb and GPIIIa crossreacted with rabbit polyclonal antibodies against human GPIIb and GPIIIa. 3. Species differences in glycosylation of GPs were shown using various lectins. 4. Molecular mass of rat major GPs was determined by SDS-PAGE (unreduced, reduced, kDa): GPIb (200, 166/26), GPIIb (140, 120/32) and GPIIIa (96, 106). 5. Isoelectric points of rat GPIIb and GPIIIa are shifted to the alkaline region as compared to human related GPs.

[Alloimmune neonatal thrombocytopenia due to anti-P1A1 thrombocyte antibodies]. / Aloimunní novorozenecká trombocytopenie zpusobená anti-P1A1 trombocytární protilátkou.

The authors describe three patients with neonatal thrombocytopenia where they detected in the maternal serum, using the immunofluorescence and immunoenzymatic test, antibodies against thrombocytes and thus confirmed the alloimmune nature of thrombocytopenia. By molecular characterization of the immunoreactivity of the thrombocytic antibody in the mother of neonate no. 1 evidence was provided that the antibody is against glycoprotein IIIa which carried antigen P1A1. In the reference laboratory in Amsterdam the anti-P1A1 specificity of this thrombocytic antibody was confirmed. The authors obtained and tested the first type anti-P1A1 serum in Czechoslovakia which can serve to detect P1A1 antigen negative subjects.