Blood PLENARY PAPERS

Dynamic imaging of fibrin network formation correlated with other measures of polymerization

Chernysh, I. N., Weisel, J. W. — 2008-05-08

Using deconvolution microscopy, we visualized in real time fibrin network formation in the hydrated state. Individual mobile fibers were observed before the gel point determined by eye. After gelation, an initial fibrin network was seen, which evolved over time by addition of new fibers and elongation and branching of others. Furthermore, some fibers in the network moved for a time. We quantified network formation by number of branch points, and longitudinal and lateral growth of fibers. Eighty percent of branch points were formed, and 70% of all fibers reached their maximum length at the gel point. In contrast, at the gel point, fiber diameter, measured as fluorescence intensity, was less than 25% and turbidity was less than 15% of the maximum values of the fully formed clot. The cumulative percentage of fibers reaching their final length and the number of branch points attained maximum values at 60% of maximum turbidity. Lateral fiber growth reached a plateau at the same time as turbidity. Measurements of clot mechanical properties revealed that the clots achieved maximum stiffness and minimum plasticity after the structural parameters reached their maxima. These results provide new information on the relative time sequence of events during fibrin network formation.

Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice

2008-04-08

An acquired somatic mutation in the JAK2 gene (JAK2-V617F) is present in the majority of patients with myeloproliferative disorders (MPDs). Several phenotypic manifestations (polycythemia vera [PV], essential thrombocythemia [ET], and primary myelofibrosis) can be associated with the same mutation. We generated JAK2-V617F transgenic mice using a human JAK2 gene with the sequences encoding the kinase domain placed in the inverse orientation and flanked by antiparallel loxP sites. Crossing mice of one transgenic line (FF1) with transgenic mice expressing Cre-recombinase under the control of the hematopoiesis specific Vav promoter led to expression of JAK2-V617F that was lower than the endogenous wild-type Jak2. These mice developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia. Induction of the JAK2-V617F transgene with the interferon-inducible MxCre resulted in expression of JAK2-V617F approximately equal to wild-type Jak2 and a PV-like phenotype with increased hemoglobin, thrombocytosis, and neutrophilia. Higher levels of JAK2-V617F in mouse bone marrow by retroviral transduction caused a PV-like phenotype without thrombocytosis. These data are consistent with the hypothesis that the ratio of mutant to wild-type JAK2 is critical for the phenotypic manifestation. A similar correlation was also found in patients with MPD.

Rac1 is essential for intraembryonic hematopoiesis and for the initial seeding of fetal liver with definitive hematopoietic progenitor cells

2008-03-24

Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesonephros are hypothesized to colonize the fetal liver, but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here, we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach, we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1, there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos, while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos, culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5, Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired inter-action with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis.