Maturation of human monocyte-derived dendritic cells studied by microarray hybridization.

We compared the transcript profiles of human myeloid immature dendritic (IDC) cells and mature dendritic cells (MDC) by hybridization of cell-derived cDNA to DNA probes immobilized on microarrays. The microarrays contained probes for 4110 known genes. We report maturation-dependent changes in transcription of clusters of differentiation, cytokines, cytokine receptors, chemokines, chemokine receptors, neuropeptides, adhesion molecules, and other genes. We identified 1124 transcripts expressed in IDC and 1556 transcripts expressed in MDC. Maturation increased the levels of 291 transcripts twofold or more and reduced the levels of 78 transcripts to one-half or less than in IDC. We identified a concerted maturation-stage-dependent transcription of the variable chains of the members of the gamma-chain-cytokine receptor family IL-4R, IL-7R, and IL-15R. Also, we found the reversal of the ratio of transcripts for galectin-3 and galectin-9 upon maturation. We identified maturation-dependent changes in the levels of transcripts for numerous genes encoding proteins previously undetected in dendritic cells such as indoleamine 2,3-deoxygenase, Epstein-Barr virus induced protein 3 and kinesin-2. Moreover, MDC transcribed and translated insulin like growth factor-1 receptor, transforming growth factor alpha, and neuropeptide Y.

Structural model of porcine factor VIII and factor VIIIa molecules based on scanning transmission electron microscope (STEM) images and STEM mass analysis.

Porcine plasma factor VIII (fVIII) molecules are heterodimers composed of a 76,000-mol wt light chain (-A3-C1-C2) and a heavy chain ranging in molecular weight from 82,000 (A1-A2) to 166,000 (A1-A2-B). Proteolytic activation of fVIII by thrombin results in fVIIIa heterotrimers lacking B domains (A1, A2, A3-C1-C2). In this study, immunoaffinity purified fVIII was further fractionated by mono S or mono Q chromatography to prepare heterodimers containing a light chain and an A1-A2-B heavy chain (fVIII 166/76) or an A1-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STEM) images of fVIII 166/76 indicated that heterodimers (mass 237 +/- 20 kD) had irregularly globular core structures 10-12 nm across, and frequently displayed a diffuse, occasionally globular to ovoid satellite structure extending 5-14 nm from the core, and attached to it by a thin stalk. Factor VIII 82/76 molecules (mass 176 +/- 20 kD) had the same core structures as fVIII 166/76 molecules, but lacked the satellite structure. These findings indicate that A1-A2 domains of heavy chains and the light chains of the fVIII procofactor molecule are closely associated and constitute the globular core structure, whereas the B domainal portion of heavy chains comprises the peripheral satellite appendage. Factor VIII core structures commonly displayed a finger-like projection near the origin of the B domainal stalk that was also a consistent feature of the free heavy chains (mass 128-162 kD) found in fVIII 166/76 preparations. Factor VIII light chain monomers (mass, 76 +/- 16 kD) were globular to c-shaped particles 6-8 nm across. These chains commonly possessed a v-shaped projection originating from its middle region, that could also be observed at the periphery of fVIII core molecules. Factor VIIIa preparations contained heterotrimers (mass 162 +/- 13 kD) that had the same dimensions as fVIII core structures, lacked the B domainal appendage, and sometimes possessed the same core features as fVIII molecules. Molecular species corresponding to heterodimers (mass, 128 +/- 13 kD) and unassociated subunit chains (40-100 kD) were also observed in fVIIIa preparations, suggesting that heterotrimers have an appreciable tendency to dissociate, a phenomenon that could explain the decay of fVIIIa activity after thrombin activation of fVIII.

Transplantation of normal bone marrow into a pig with severe von Willebrand's disease.

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: the plasmatic compartment is only minimally replenished by the vWF from platelets and megakaryocytes; and the platelet vWF alone only partially corrects the abnormal tests of the hemostatic mechanism in severe vWd.

Activation of porcine factor VIII:C by thrombin and factor Xa.

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)

Internal duplication and sequence homology in factors V and VIII.

Blood coagulation factors V and VIII each serve cofactor functions with different vitamin K-dependent serine proteases of the coagulation cascade. Physical, physiologic, and kinetic data suggest analogous structures and functions for these two proteins. Proteolytically activated factor V (factor Va) is required for the efficient production of thrombin from prothrombin by factor Xa. Similarly, activated factor VIII (factor VIIIa) performs its cofactor activity with factor IXa to produce the activated form of factor X (factor Xa). The studies reported here on the sequences from the thrombin-activated and unactivated cofactors provide evidence that factor V and factor VIII are chemically related and that the structures of both cofactors involve some tandem duplication.

Coagulation factors V and VIII and ceruloplasmin constitute a family of structurally related proteins.

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 683-791. 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.

Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid.

The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino-naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.

Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein.

Partially purified preparations of porcine factor VIII:C were used to immunize mice and spleen cells from the immunized animals were fused to NS-1 mouse myeloma cells. The ability of hybrid culture fluids to bind factor VIII:C was detected with a radiolabelled, affinity-purified, human antihuman VIII:C inhibitor. Three cloned hybrid lines have been obtained that preferentially bind to VIII:C when compared to von Willebrand factor binding. Two of these monoclonal antibodies partially inhibit VIII:C coagulant activity. The third antibody does not inhibit VIII:C, but it can be used as an affinity reagent to absorb dissociated VIII:C out of solution. Active coagulant can be recovered by elution in 50% ethylene glycol. The VIII:C obtained has a specific activity of 6 units/micrograms based on absorbance measurements. When analyzed on SDS gels, the unactivated VIII:C contains 3 bands of apparent molecular weight 166,000, 130,000 and 76,000. Thrombin treatment results in a 40 fold increase in activity and cleavage to products of 76,000, 67,000 an 50,000 and small amounts of lower molecular weight peptides. EDTA inactivation of the factor VIII:C results in the separation of the 166,000 and 130,000 chains from the 76,000 chain, suggesting a Ca++ dependent noncovalent interaction among the chains.

Porcine Willebrand factor: a population of multimers.

Purified porcine Willebrand factor was analyzed by agarose-sodium DodSO4 electrophoresis. Multiple forms of the protein were found in a series of increasing molecular weights. A molecular mass calibration curve was constructed with fibrinogen (3.4 X 10(5) daltons), IgM (1 X 10(6) daltons), and glutaraldehyde-crosslinked IgM polymers (2, 3, and 4 X 10(6) daltons). As measured by this procedure, the apparent molecular weight of Willebrand factor polymers ranged from 1.1 X 10(6) to 2.1 X 10(7). Each member of the series differed from one another by approximately 1.5 to 1.9 X 10(6) daltons, indicating that members of the series were polymers of 6-mers to 8-mers of the 2.3 X 10(5) dalton subunit. Various purification procedures, used to isolate Willebrand factor active in inducing platelet aggregation, were seen to fractionate the polymers, in part, on the basis of size. The same purification procedures, when applied to procine von Willebrand plasma, failed to yield protein of molecular weight greater than 1.1 X 10(6).