Mutation at either Arg336 or Arg562 in factor VIII is insufficient for complete resistance to activated protein C (APC)-mediated inactivation: implications for the APC resistance test.

Activated protein C (APC)-mediated inactivation of factor VIII (FVIII) correlates with cleavage at either Arg336 and/or Arg562. To elucidate the APC cleavage requirements for inactivation of FVIII, APC cleavage site mutants in FVIII (R336I, R562K and R336I/R562K) were made by site-directed mutagenesis. Analysis of these FVIII mutants expressed in COS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was resistant to APC cleavage at residue 562 but not at Arg336 and the thrombin cleaved mutant R3361 was mostly resistant to APC cleavage at residue 336, but was sensitive to APC cleavage at Arg562. The double mutant R336I/R562K was mostly resistant to cleavage at residue 336 and completely resistant to cleavage at residue 562. Thus, APC cleavage of FVIII does not require a specific order of cleavage at either residue. The functional inactivation by APC was studied using partially purified preparations of FVIII expressed in Chinese hamster ovary cells. Both single mutants were inactivated at similar rates but slower than wild-type FVIII, whereas the double mutant R336I/R562K was resistant to inactivation. The ability of a commercially available APC-resistance assay kit to detect APC resistant FVIII was tested by reconstituting FVIII deficient plasma with the APC resistant mutants. Only the R336I/R562K demonstrated a reduced APC-resistance ratio, indicating that this assay can not detect the single APC cleavage site mutant of FVIII. These results suggest that APC-mediated cleavage at either Arg336 or Arg562 partially inactivate FVIII.

Characterization of a neuregulin-related gene, Don-1, that is highly expressed in restricted regions of the cerebellum and hippocampus.

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.

Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage.

Factor VIII functions as an essential cofactor in the blood coagulation cascade for the factor IXa-mediated activation of factor X. Factor VIII contains 6 tyrosine residues at positions 346, 718, 719, 723, 1664, and 1680 that are modified by post-translational sulfation. This modification is required for full factor VIII procoagulant activity. We have employed site-directed mutagenesis to identify the individual sulfated tyrosines within factor VIII that influence activity. The molecules were expressed in COS-1 monkey cells by transient transfection, and the resultant proteins were characterized. Metabolic incorporation of [35S]sulfate demonstrated that all 6 tyrosine residues are sulfated in factor VIII. Sulfation at residues 346 and 1664 was required for full activity in a factor VIII clotting assay but did not affect factor VIII activity monitored by a factor Xa generation assay. The Tyr346-->Phe and Tyr1664-->Phe mutants displayed delayed thrombin activation that correlated with delayed cleavage at residues 372 and 1689, respectively. In contrast, these mutants were efficiently activated by factor Xa. A triple Tyr to Phe mutant at residues 718, 719, and 723 displayed both reduced factor VIII clotting activity and factor Xa generation activity. Finally, a Tyr1680-->Phe mutant factor VIII displayed a 5-fold reduced affinity for von Willebrand factor. The results demonstrate that 1) sulfation at tyrosine residues 346 and 1664 increases factor VIII activity by increasing the rate of thrombin activation and cleavage; 2) sulfation at tyrosine residues 718, 719, and 723 increases the intrinsic activity of factor VIIIa; and 3) sulfation at tyrosine residue 1680 increases the affinity for vWF. In addition, the results implicate that thrombin interacts with three distinct sites within factor VIII, two of which are required for proteolytic activation. The results demonstrate that the six sites of tyrosine sulfation modulate factor VIII activity through different mechanisms.

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers.

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain. To do this analysis we chose to examine the relative Ig H chain V region gene usage of pre-B cell lines derived from (C57BL/6 x BALB/c)F1 fetal liver cells by Southern blot analysis. We found a high frequency of Vh-gene rearrangements (77% of the alleles had VDJ rearrangements) and these rearrangements occurred to Vh-genes throughout the Vh locus and were not confined to the D-proximal Vh-genes as has been previously observed with lines from other mouse strains. The Vh-gene usage pattern is similar on both alleles indicating that at least one of the determinants of which Vh-gene is used is trans-acting and acts similarly on each allele. Furthermore, one allele, Ighb (donated by the C57BL/6 parent), rearranged Vh-genes more frequently than the other allele, Igha (donated by the BALB/c parent) suggesting that one of the determinants of Vh-gene rearrangement may be acting in an allele-specific manner.