X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

Telomerase activity in normal and malignant hematopoietic cells.

Bone marrow and peripheral blood leukocytes from 19 leukemia patients were found to contain telomerase activity detectable by a PCR-based assay. Telomerase was also detectable in nonmalignant bone marrow and peripheral blood leukocytes from normal donors, including fractions enriched for granulocytes, T lymphocytes, and monocytes/B cells. Semiquantitative comparison revealed considerable overlap between telomerase activities in samples from normal subjects and leukemia patients, confounding evaluation of the role of telomerase in this disease. These data indicate that human telomerase is not restricted to immortal cells and suggest that the somatic expression of this enzyme may be more widespread than was previously inferred from the decline of human telomeres.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.