Islet and hepatocyte transplantation are associated with tissue factor-dependent activation of coagulation which elicits instant blood mediated inflammatory reaction, thereby contributing to a low rate of engraftment. The aim of this study was i) to evaluate the procoagulant activity of human adult liver-derived mesenchymal progenitor cells (hALPCs), ii) to compare it to other mesenchymal cells of extra-hepatic (bone marrow mesenchymal stem cells and skin fibroblasts) or liver origin (liver myofibroblasts), and iii) to determine the ways this activity could be modulated. Using a whole blood coagulation test (thromboelastometry), we demonstrated that all analyzed cell types exhibit procoagulant activity. The hALPCs pronounced procoagulant activity was associated with an increased tissue factor and a decreased tissue factor pathway inhibitor expression as compared with hepatocytes. At therapeutic doses, the procoagulant effect of hALPCs was inhibited by neither antithrombin activators nor direct factor Xa inhibitor or direct thrombin inhibitors individually. However, concomitant administration of an antithrombin activator or direct factor Xa inhibitor and direct thrombin inhibitor proved to be a particularly effective combination for controlling the procoagulant effects of hALPCs both in vitro and in vivo. The results suggest that this dual antithrombotic therapy should also improve the efficacy of cell transplantation in humans.

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

See also Randriamboavonjy V, Fleming I. Energy and motion: AMPKa1 and its role in platelet activation. This issue, pp 970-2.Summary. Background: Platelet activation requires sweeping morphologic changes, supported by contraction and remodeling of the platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets. Objective: To determine whether AMPK is activated during platelet aggregation and contributes to the control of cytoskeletal targets. Results: We found that AMPK-a1 was mainly activated by thrombin, and not by other platelet agonists, in purified human platelets. Thrombin activated AMPK-a1 ex vivo via a Ca 2+ / calmodulin-dependent kinase kinase b (CaMKKb)-dependent pathway. Pharmacologic inhibition of CaMKKb blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLCs), cofilin, and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeletal contraction and polymerization. Platelets isolated from mice lacking AMPK-a1 showed reduced aggregation in response to thrombin, and this was associated with defects in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show, for the first time, that the AMPK pathway is activated in platelets of patients undergoing major cardiac surgery, in a heparin-sensitive manner. Conclusion: AMPK-a1 is activated by thrombin in human platelets. It controls the phosphorylation of key cytoskeletal targets and actin cytoskeletal remodeling during platelet aggregation.

The intron 22 inversion found in up to 50% of severe hemophilia A patients results from a recombination between three intron 22 homologous copies (int22h). This study evaluated the implication of these copies in the formation of extended duplications comprising exons 1-22 of the factor 8 (F8) gene and their association with hemophilia and mental retardation. Two hemophilic patients with moderate and severe phenotypes and a third nonhemophilic patient with developmental delay were studied. All exhibited a duplication of F8 gene exons 1-22 identified by multiplex ligation-dependent probe amplification along with abnormal patterns on Southern blotting and unexpected long-range PCR amplification. Breakpoint analysis using array comparative genomic hybridization was performed to delimit the extent of these rearrangements. These duplications were bounded on one side by the F8 intragenic int22h-1 repeat and on the other side by extragenic int22h-2 or int22h-3 copies. However, the simultaneous identification of a second duplication containing F8 gene exons 2-14 for the moderate patient and the classical intron 22 inversion for the severe patient are considered in this study as the genetic causal defects of hemophilia. This study shows that the well-known int22h copies are involved in extended duplications comprising F8 gene exons 1-22. These specific duplications are probably not responsible for hemophilia and intellectual disability, but should be carefully considered in genetic counseling, while continuing to investigate the causal mutation of hemophilia.

Background: Several hemostatic abnormalities have been described in hypothyroidism, such as modification of coagulation proteins and bleeding tendency. Although thyroid hormone deficiency is considered to be responsible for these changes, the underlying mechanisms have not yet been established. Objective: To evaluate the respective influence of peripheral thyroid hormones (free thyroxine) and TSH on blood clotting by assessing coagulation parameters in patients with a history of total thyroidectomy for thyroid cancer, under three different conditions: induced hypothyroidism, euthyroid state, and following recombinant human TSH (rhTSH) administration. Methods: Coagulation parameters (platelet count, fibrinogen, international normalized ratio, prothrombin time, thrombin time, activated partial thromboplastin time (APTT), factor VIII activity ((FVIII:C), as well as von Willebrand factor antigen (VWF:Ag) and VWF activity using collagen binding assay (VWF:CBA)) were measured in patients with severe hypothyroidism following withdrawal of thyroid hormone replacement therapy, and in the same patients with euthyroidism after restoring replacement treatment (group A), and before and after administering rhTSH (group B). Results: FVIII:C, VWF:Ag, and VWF:CBA were significantly decreased (P!0.001), whereas APTT was significantly increased (P!0.001) in patients with severe hypothyroidism compared with patients in the euthyroid state. No changes in clotting parameters were observed in patients who received rhTSH therapy.Conclusion: This prospective study shows that severe short-term hypothyroidism is associated with significantly lower levels of VWF:Ag, VWF:CBA, and FVIII:C. Administration of exogenous TSH has no effect on coagulation parameters. These findings suggest that thyroid hormone deficiency is likely to be the main cause of coagulation alterations in patients with hypothyroidism.