Rotation thromboelastometry analysis of clot formation and fibrinolysis in severe thrombocytopenia: effect of fibrinogen, activated prothrombin complex concentrate, and thrombin- activatable fibrinolysis inhibitor.

INTRODUCTION: Bleeding symptoms in severe thrombocytopenia range from mild to severe. The aim of this in vitro study was to improve blood clotting and protect against fibrinolysis in reconstituted severe thrombocytopenia blood. METHODS: Thrombocytopenia [(16 ± 4) × 10(6) /mL] was created by high-speed centrifugation of normal blood with subsequent mixing plasma with packed cells. The blood samples were subjected to clotting by CaCl2 and tissue factor and to fibrinolysis by the addition of tissue plasminogen activator. Blood was spiked with fibrinogen, activated prothrombin complex concentrate (FEIBA), thrombin activatable fibrinolysis inhibitor (TAFI), or their combinations. To mimic the situation that may occur in patients subjected to massive transfusion of plasma substitutes, blood was diluted by 40% of TRIS/saline buffer. Clotting time (CT), α-Angle, maximum clot firmness (MCF), and lysis onset time (LOT) were evaluated using rotation thromboelastometry. RESULTS: Spiking thrombocytopenia blood with FEIBA led to reduction of CT. Fibrinogen and FEIBA enhanced α-Angle and MCF both in the absence and in the presence of tPA. LOT values were prolonged by TAFI and to less extent by FEIBA. Dilution of thrombocytopenia blood was followed by reduction of α-Angle and MCF compared to nondiluted blood which partly reversed by either fibrinogen or FEIBA being higher using fibrinogen and FEIBA together. Clot strength was enhanced, and fibrinolysis was inhibited by TAFI. CONCLUSION: The results of this study suggest that combined spiking of blood with fibrinogen and FEIBA may be enough to correct the clot formation disorder in severe thrombocytopenia, whereas in thrombocytopenia and blood dilution, additive inhibition of fibrinolysis may be needed.

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the ß-propeller (Asn2Asp) disrupts a calcium binding site in blade 6.

BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbß(3). OBJECTIVE: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the ß-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbß(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT ß(3). Although the αIIbß(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbß(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbß(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbß(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbß(3), αVß(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVß(3) to calcium chelation compared with αIIbß(3). CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the ß-propeller for intracellular trafficking of αIIbß(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

Testing agonist-induced platelet aggregation by the Impact-R [Cone and plate(let) analyzer (CPA)].

The Impact-R [Cone and plate(let) analyzer (CPA)] is useful to assess platelet adhesion in different diseases and to monitor antiplatelet therapy. The purpose of the present study was to adapt this system to test agonist-induced platelet aggregation. Blood samples were tested by light transmission platelet aggregometry (LTA), Impact-R regular test and Impact-R agonist-response test. In the latter, samples were pre-incubated for 1 min with an agonist leading to platelet activation, micro-aggregates formation and reduced adhesion. Impact-R regular test of ten healthy volunteers demonstrated platelet adhesion (surface coverage, SC) of 11.2 +/- 2.6% while LTA induced by ADP, ristocetin, epinephrine, collagen and arachidonic acid (AA) yielded maximal aggregation (81% to 93%). In the Impact-R agonist-response test, SC was reduced to 2.2 +/- 1.0%, 1.2 +/- 0.9%, 2.3 +/- 1.0%, 2.2 +/- 0.8% and 2.4 +/- 0.4%, respectively. Prostaglandin E(1) treatment weakened SC reduction in response to ADP and epinephrine (SC of 8.8 +/- 1.8% and 9.5 +/- 2.0%, respectively). Inhibition of P2Y(12) receptor with 2MeSAMP resulted in a dose-dependent decrease in maximal aggregation in the ADP-induced test, which inversely correlated to SC in the Impact-R ADP-response test. The Impact-R agonist-response tests detected aggregation defects in patients with storage pool disease, severe von Willebrand disease and epinephrine response deficiency, and may be useful to assess the effect of different agonists on platelet aggregation.

Glow curve analysis of composite peak 5 in LiF:Mg,Ti (TLD-100) using optical bleaching, thermal annealing and computerised glow curve deconvolution.

The relative intensity of glow peak 5a in the composite glow peak 5 of LiF:Mg,Ti (TLD-100) is very weak following gamma irradiation, and has been estimated at approximately 0.1 of the intensity of peak 5. Typical glow curve analysis using computerised glow curve deconvolution with unconstrained variation of the peak shape parameters, yields values of the relative intensity of glow peak 5a varying from 0 to 15%. Due to the potential of peak 5a to fulfil the criteria of a quasi-tissue-equivalent nanodosemeter which estimates quality factor, considerable efforts have been invested in ancilliary techniques to improve the reliability of the estimation of the intensity of peak 5a. Optical bleaching and thermal annealing techniques were used to obtain single-peak glow curves consisting of peak 4 only and peak 5 only. A multi-stage CGCD protocol was then constructed using these peak shape parameters for peaks 4 and 5, which allows more accurate estimation of the relative intensity of peak 5a. Following 60Co irradiation of ten chips to a dose level of 1 Gy, the technique yields a relative intensity of 0.08 +/- 0.008 (1 SD).

The composite structure of peak 5 in the glow curve of LiF:Mg,Ti (TLD-100): confirmation of peak 5a arising from a locally trapped electron-hole configuration.

The hypothesis that glow peak 5a arises from localised e-h capture is confirmed by the following experimental observations: (i) The high conversion efficiency (CE) (CE5a-->4 = 3 +/- 0.5) of peak 5a to peak 4 (a hole-only trap) deduced from detailed Im-Tstop optical bleaching studies at 310 nm compared to the much lower CE of peak 5 (an electron-only trap) (CE5-->4 = 0.0026+/-0.012). (ii) The lack of an increase in the sensitivity of glow peak 5a following 2.6 MeV and 6.8 MeV He ion irradiation in 'sensitised' material compared to the factor two increase in the sensitivity of peak 5; (S/S0)5a = 0.86+/-0.12, compared to (S/S0)5 = 2.0+/-0.2. (iii) The late entry into saturation of the 2.6 MeV and 6.8 MeV He ion TL-fluence response curves for peak 5a compared to peak 5 in sensitised and normal material resulting in the following values for the track radial saturation parameter: (r50)5a = 100+/-20) Angstroms compared to (r50)5 = 380+/-30 Angstroms. (iv) The low value of 0.1 for the 'track-escape' parameter of peak 5a deduced from the Extended Track Interaction Model analysis of He ion TL fluence response compared to order of magnitude greater values for peaks 5 and 5b.

Localised and delocalised optically induced conversion of composite glow peak 5 in LiF:Mg,Ti (TLD-100) to glow peak 4 as a function of postirradiation annealing temperature.

The composite structure of glow peak 5 in LiF:Mg,Ti (TLD-100) has been investigated using optical bleaching by 310 nm (4 eV) light. The glow peak conversion efficiency of peak 5a (Tm = 187 degrees C) to peak 4 traps is very high at a value of 3+/-0.5 (1 SD) whereas the glow peak conversion efficiency of peak 5 (Tm = 205 degrees C) to peak 4 traps is 0.0026+/-0.0012 (1 SD). The high conversion efficiency of peak 5a to peak 4 arises from direct optical ionisation of the electron in the electron-hole pair. leaving behind a singly-trapped hole (peak 4), a direct mechanism, relatively free of competitive mechanisms. Optical ionisation of the 'singly-trapped' electron (peak 5), however, can lead to peak 4 only via multi-stage mechanisms involving charge carrier transport in the valence and conduction bands, a mechanism subject to competitive processes. The conduction/valence band competitive processes lead to the factor of one thousand decrease in the conversion efficiency of peak 5 compared to peak 5a.

Implantation of rat insulinoma cell line into cyclosporine treated rats. Effect of the in vivo environment on beta-cell specific gene expression.

BACKGROUND: Transplantation of engineered beta cell-derived lines is a promising modality for cell-based therapy of diabetes mellitus. The in vivo environment and antirejection and other medications may have significant effects on the differentiation and proliferation of the transplanted beta cells, thus affecting their function. The effect of the in vivo environment on expression of genes encoding proteins involved in insulin production, secretion, and glucose sensing were analyzed in the RIN 104638 cell line. METHODS: RIN 104638 cells, were used for s.c. implantation in cyclosporine treated rats and for parallel in vitro culture. The differential expression of the insulin, PDX-1, GLUT-2, and glucokinase genes were assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: The in vivo environment of cyclosporine-treated rats, preserved most of the differentiated characteristics of the implanted cells. Insulin and glucokinase gene expression were maintained at high levels, although GLUT-2 expression decreased. This was in contrast to the substantial decrease of all the three genes expression when cultured in vitro. Cyclosporine treatment reduced insulin and GLUT-2 gene expression in in vitro culture. CONCLUSIONS: Beta cell implantation in cyclosporine-treated rats induces alteration in expression of genes pivotal to insulin production and secretion and the glucose sensing abilities. The normal in vivo environment improves the implanted b cell function by increasing the insulin gene expression and content. Furthermore, it reverses some of the dedifferentiating changes caused by the in vitro culture. This may have a positive effect on the therapeutic efficiency of this cell line.

Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia.

Insulin gene expression is restricted to islet beta cells of the mammalian pancreas through specific control mechanisms mediated in part by specific transcription factors. The protein encoded by the pancreatic and duodenal homeobox gene 1 (PDX-1) is central in regulating pancreatic development and islet cell function. PDX-1 regulates insulin gene expression and is involved in islet cell-specific expression of various genes. Involvement of PDX-1 in islet-cell differentiation and function has been demonstrated mainly by 'loss-of-function' studies. We used a 'gain-of-function' approach to test whether PDX-1 could endow a non-islet tissue with pancreatic beta-cell characteristics in vivo. Recombinant-adenovirus-mediated gene transfer of PDX-1 to the livers of BALB/C and C57BL/6 mice activated expression of the endogenous, otherwise silent, genes for mouse insulin 1 and 2 and prohormone convertase 1/3 (PC 1/3). Expression of PDX-1 resulted in a substantial increase in hepatic immunoreactive insulin content and an increase of 300% in plasma immunoreactive insulin levels, compared with that in mice treated with control adenovirus. Hepatic immunoreactive insulin induced by PDX-1 was processed to mature mouse insulin 1 and 2 and was biologically active; it ameliorated hyperglycemia in diabetic mice treated with streptozotocin. These data indicate the capacity of PDX-1 to reprogram extrapancreatic tissue towards a beta-cell phenotype, may provide a valuable approach for generating 'self' surrogate beta cells, suitable for replacing impaired islet-cell function in diabetics.