Jaroslav G. Vostal

Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria

We analysed endothelial cell membrane microparticles (ECMP) in the peripheral blood of patients with paroxysmal nocturnal haemoglobinuria (PNH) (n = 9), aplastic anaemia (AA) (n = 10), sickle cell disease (SCD) (n = 8), and healthy donors (HD) (n = 11). There was no clinically manifested thrombosis in the PNH or AA group, except one cured thrombophlebitis (PNH), while all SCD patients had a history of vaso‐occlusive crises. We used three‐colour flow cytometry with blood cell‐specific antibodies and antibodies to endothelial antigens CD105 and CD144. Phosphatidylserine‐positive microparticles were detected using the annexin V‐binding (AVB) assay. The population of CD105+AVB+ ECMP was significantly (P < 0·05) higher in SCD (median: 0·568 × 109/l; 25–75th percentile range: 0·351–0·976 × 109/l) and PNH (0·401 × 109/l ; 0·19–0·441 × 109/l) patients when compared with AA (0·122 × 109/l; 0·061–0·172 × 109/l) or HD (0·180 × 109/l; 0·137–0·217 × 109/l) group. Even more pronounced differences were observed in ECMP exhibiting a marker of inflammatory stimulation CD54 (CD105+CD54+). Similarly, ECMP that exhibited endothelial specific and proteolysis‐sensitive antigen CD144 were increased in SCD and PNH, but not in AA. Elevated CD54+ ECMP may reflect the inflammatory status of endothelial cells in SCD and PNH, while CD144+ ECMP could indicate continuous endothelial stimulation and/or injury. Analysis of circulating ECMP appears promising to provide useful information on the status of the vascular endothelium in PNH and SCD.

Toxicity of a first-generation adenoviral vector in rhesus macaques

We constructed a first-generation adenovirus vector (AVC3FIX5) that we used to assess the rhesus macaque as a nonhuman primate model for preclinical testing of hemophilia B gene therapy vectors. Although we succeeded in our primary objective of demonstrating expression of human factor IX we encountered numerous toxic side effects that proved to be dose limiting. Following intravenous administration of AVC3FIX5 at doses of 3.4 x 10(11) vector particles/kg to 3.8 x 10(12) vector particles/kg, the animals in our study developed antibodies against human factor IX, and dose-dependent elevations of enzymes specific for liver, muscle, and lung injury. In addition, these animals showed dose-dependent prolongation of clotting times as well as acute, dose-dependent decreases in platelet counts and concomitant elevation of fibrinogen and von Willebrand factor. These abnormalities may be caused by the direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in IL-6, a key regulator of the acute inflammatory response. The rhesus macaque may be a useful animal model in which to evaluate mechanisms of adenovirus toxicities that have been encountered during clinical gene therapy trials.

Peroxynitrite-induced tyrosine nitration and phosphorylation in human platelets.

Peroxynitrite (ONOO-) induces nitration of tyrosine residues and inhibits tyrosine phosphorylation in cell free systems. We investigated the effect of peroxynitrite on protein tyrosine nitration and phosphorylation in resting or thrombin-activated platelets. Peroxynitrite (150 microM) rapidly induced tyrosine nitration of 187, 164, 113, 89, and 61 kDa proteins in gel-filtered platelets which persisted up to 4.5 h. Repeated exposure of platelets to peroxynitrite produced increasing levels of nitration. Peroxynitrite also rapidly increased tyrosine phosphorylation of 120, 117, 95, 80-85, and 70 kDa platelet proteins, but this decreased by 5 min. The same pattern of tyrosine phosphorylation, but with higher intensity, was induced by thrombin in control platelets. Pretreatment of platelets with peroxynitrite decreased thrombin-induced tyrosine phosphorylation at 0.05 and 1 U/ml thrombin but not at 2 U/ml thrombin. Platelet activation responses such as P-selectin expression, serotonin secretion, and aggregation were also decreased by peroxynitrite treatment at low thrombin concentrations. Peroxynitrite exposure and tyrosine nitration decreased platelet sensitivity to thrombin but did not absolutely prevent tyrosine phosphorylation and other platelet responses.

Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin

BackgroundElevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail.ResultsOvernight stimulation of HUVEC with either LPS or TNFα, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 μM camptothecin, documented by 60–70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (~80,000 MP/103 cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3–1 μm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.ConclusionsCamptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.

Scrapie Infectivity in Hamster Blood Is Not Associated with Platelets

ABSTRACT The infectivity of hamster scrapie strain 263K was measured in platelets isolated from blood pooled from six hamsters with clinical scrapie. The total number of infectious doses present in the blood pool was 220, out of which only 3.5 infectious doses were associated with platelets. A larger proportion of the total infectivity was recovered from the mononuclear leukocyte fraction. This result indicates that platelets are not the source of blood-borne infectivity in transmissible spongiform encephalopathy-infected hamsters.

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.

Cellular prion protein is expressed on endothelial cells and is released during apoptosis on membrane microparticles found in human plasma.

BACKGROUND: Blood and plasma of animals experimentally infected with transmissible spongiform encephalopathies (TSEs) can transmit TSE infection by transfusion. A conformational isoform of prion protein (PrPsc) is believed to be the TSE‐infectious agent that propagates by converting the cellular prion protein (PrPc) to additional molecules of PrPsc. In orally infected animals, PrPsc accumulates in intestinal endothelial cells. In blood, two thirds of PrPc resides in plasma, but its source is not known.

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

The surface expression of prion protein (PrPC) 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 PrPC occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37°C. In comparison, PrPC on the surface of platelets, activated at 22°C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrPC and P‐selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrPC was translocated from internal granules to the plasma membrane during activation, as is P‐selectin. Platelet PrPC 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 PrPC.

FDA approach to evaluation of pathogen reduction technology

athogen reduction (PR) is a novel approach to decreasing pathogen transmission by blood transfusion. It has the potential to inactivate a broad range of pathogens that could be found in donated blood products and thus it would complement existing methods to prevent or decrease the presence of pathogens. The current methods include donor selection, skin disinfection, diversion of initial collection, and testing for specific agents. The concept of PR holds great promise. Ideally, PR methods should inactivate all pathogens found in blood products, not damage the transfusion product, and be safe to be administered to all patients. In reality, PR methodology is in its infancy and has yet to reach its true potential. In their current form, PR treatments are unlikely to sterilize the transfusion product. This results from large loads of certain pathogens that could overwhelm the capacity of the treatment, resistant forms of pathogens, inaccessible pathogens due to interference from storage bag geometry, poor light energy delivery due to interfering substances, and the potential for human error during processing. The PR methods are not strictly pathogen specific and produce collateral damage to the transfusion products that is often evident by the decreased retention of these products in circulation. In addition, the toxicity of PR chemicals and their metabolites and adducts may not be realized until a large-scale patient population is exposed to them. The FDA will evaluate PR treatments for their efficacy in decreasing pathogens in blood products, their effect on the transfusion product, and their safety to the recipient of treated products.

Intravenous administration of replication‐incompetent adenovirus to rhesus monkeys induces thrombocytopenia by increasing in vivo platelet clearance

Summary.  A replication‐incompetent adenovirus vector was administered to rhesus macaques at 1, 3 and 6 × 1012 particles/kg doses to investigate its toxicity. Platelet count decrements of 28%, 82% and 90%, respectively, were observed, with corresponding platelet half‐lives of 69·0, 25·2 and 22·2 h (compared with 111 h in untreated animals). The platelet decline was equivalent for all three doses for 8 h, and platelet count recovery began as early as 8 h after infusion for low‐dose recipients, or as late as 24 h for the medium and high dose recipients. These observations suggest that thrombocytopenia is a saturable, reversible consumptive process.