IntroductionImatinib mesylate (Gleevec; Novartis, Basel, Switzerland) is a reversible tyrosine kinase inhibitor effective in treatment of chronic myelogenous leukemia 1 (CML), gastrointestinal stromal tumors, 2,3 eosinophilic disorders, 4 and systemic mast cell disease 5 and has been tried in several other diseases. 6,7 The drug binds preferentially to adenosine triphosphate (ATP)-binding sites of the c-kit protooncogene product, platelet-derived growth factor receptor (PDGF-R), and Abelson kinase (c-ABL) [8][9][10] impeding the ensuing signal transduction (for a review, see Savage and Antman 11 ). Inhibition of the ABL portion of the BCR-ABL fusion protein, pathognomonic of CML, 12 is the basis of imatinib mesylate efficacy in inducing hematologic and cytogenetic remissions in CML. 1,13 Inhibition of c-KIT is considered critical for imatinib mesylate effects in gastrointestinal stromal tumors 14 and mast cell disease. 5 In our efforts to develop immunotherapy for CML, 15,16 we studied the effects of imatinib mesylate on the development of CML-specific immunity on administration of autologous mature dendritic cells (DCs). Consequently, we measured the effects of imatinib mesylate on in vitro correlates of immunity. 17 At concentrations of 5 M, achieved in the serum of patients on the standard therapeutic dose of 400 mg/d, 1 the drug was cytostatic, it arrested transit into the S phase of the cell cycle, and it impeded in vitro proliferation of human normal and leukemic T cells. We analyzed the effects of imatinib mesylate on the activity of intracellular signal transduction molecules associated with the control of T-cell function. The drug diminished phosphorylation of Lck, ERK 1/2, and Rb proteins and reduced the levels of cyclin D3 and of activated nuclear transcription factor B (NF-B). In addition, imatinib mesylate inhibited delayed-type hypersensitivity (DTH) in mice without affecting T-cell numbers, indicating that the drug inhibits T-cell response but does not induce apoptosis. Materials and methods Cells and reagentsT cells were isolated by negative immunoadsorption (Pan T kit; Miltenyi Biotec, Auburn, CA), pooled from 3 or more normal donors and cryopreserved until use. On thawing, the cells were incubated in X-VIVO 15 medium (BioWhittaker, Walkersville, MD) supplemented with 1.0% human AB serum (Sigma, St Louis, MO), in a humidified atmosphere of 5.0% CO 2 at 37°C. For experiments longer than 1 day, the medium contained 1.0% penicillin/streptomycin solution (Sigma) as well. Human acute T-cell leukemia cells Jurkat (ATCC TIB-152), acute T lymphoblastic leukemia cells CCRF-CEM (ATCC CCL-119), and hematopoietic malignant K-562 cells (ATCC CCL-243), were obtained from American Type Culture Collection (Manassas, VA) and cultured under the same conditions. Allogeneic DCs were derived from CD14 ϩ cells and cultured with T cells as previously described. 16 Imatinib mesylate (Novartis) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM. The stock solution was stored at Ϫ20°C until use,...

The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 X 10(6) M-1 s-1 and 1.1 X 10(6) M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer searches of the National Biomedical Research Foundation protein and nucleic acid sequence data bases using the NH2 terminus of the bovine factor Va 94-kilodalton heavy chain, the NH2 terminus of the 74-kilodalton factor Va light chain, and an internal 98-residue segment of porcine factor VIII revealed that both bovine factor V and porcine factor VIII are statistically homologous to human ceruloplasmin. The NH2-terminal segment of bovine factor Va heavy chain is homologous to three segments of ceruloplasmin sequence starting at residues 1, 351, and 713; the NH2-terminal sequence of bovine factor Va light chain is homologous to the same human ceruloplasmin sequence segments beginning at residues 1, 349, and 711. The longer porcine factor VIII sequence is homologous to three segments of human ceruloplasmin, residues 1-77, 400-433, and 683-191. These data indicate that factor V, factor VIII, and ceruloplasmin comprise a group of evolutionarily linked protein structures that possibly resulted from multiplication of ancestral precursor genes.Factor V and factor VIII each function as cofactors for two vitamin K-dependent serine proteases in the blood coagulation cascade (1). Bovine factor V (BFV) is a single chain glycoprotein of mass 330 kDa, which is cleaved to an active two-polypeptide form (factor Va) consisting of a 94-kDa heavy chain complexed to a 74-kDa light chain via Ca2+-dependent forces (2). The 94-kDa peptide of bovine factor Va corresponds to the NH2-terminal region of factor V; the 74-kDa peptide corresponds to the COOH-terminal region of factor V (3, 4). Factor Va functions in coagulation as a receptor on phospholipid vesicles (5) and platelets (6, 7) for the serine protease factor Xa. This complex of factor Xa, factor Va, a membrane surface, and calcium ion catalyzes the cleavage of the zymogen prothrombin to thrombin. As isolated, porcine factor VIII (PFVIII) is a two-chain structure composed of 160-and 76-kDa peptides whose association appears to depend on the presence of Ca2+ ion (8). COOHterminal cleavage of the 160-kDa heavy chain gives rise to an altered form of the two-chain cofactor consisting of a 130-kDa modified heavy chain associated with the 76-kDa light chain. Activation of factor VIII with thrombin gives rise to further loss of the COOH segment of the heavy chain, yielding a heavy chain-derived polypeptide of 82 kDa associated with a light chain-derived fragment of 69 kDa. In coagulation, activated factor VIII (factor VIIa), in the presence of Ca2+ and phospholipid, appears to function as a cofactor for the seine protease factor IXa in the activation of factor X to factor Xa. Recently EXPERIMENTAL PROCEDURES Protein sequences were determined by using an Applied Biosystems gas-phase sequencer using PFVIII isolated as described by Fass et al. (8) and using BFV isolated as described by Nesheim et al. (11). Two sequences for BFV were used for searching the Atlas of Protein Sequence and Structure (10)-namely, the 29 NH2-terminal residues of the 94-kDa heavy chain and the 25...

Bone marrow from a normal male pig was transplanted into a related female pig with severe homozygous von Willebrand's disease (vWd). After engraftment the circulating leukocytes were of the male karyotype, and the platelets were strongly positive for von Willebrand factor (vWF) by indirect immunofluorescence. The average level of vWF was 1.96 U/dl and of ristocetin cofactor was 2.8 U/dl. The ear immersion bleeding time before transplantation was consistently more than 15 min and afterwards varied between 5 min and more than 15 min. Transfused vWF corrected the bleeding time at a level of 10 U/dl, which is lower than that required for a von Willebrand pig. We concluded that: (a) the plasmatic compartment is only minimally replenished by the vWF from platelets and megakaryocytes; and (b) the platelet vWF alone only partially corrects the abnormal tests of the hemostatic mechanism in severe vWd.

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