David N. Fass

Resistance to Arteriosclerosis in Pigs with von Willebrand's Disease: SPONTANEOUS AND HIGH CHOLESTEROL DIET-INDUCED ARTERIOSCLEROSIS

The aortas of 11 pigs (aged 1-3 yr) with homozygous von Willebrands disease (vWd) were compared with those of 11 normal pigs of the same ages. Six of the controls exhibited multiple arteriosclerotic plaques with intimal thickening of 63-130 mum. In contrast, none of the pigs with vWd had multiple plaques, and only one had a lesion >2 mm in diameter. In a subsequent study, 3-mo-old pigs (11 controls and 7 with homozygous vWd) were placed on a 2% cholesterol diet for up to 6 mo. All of the controls developed arteriosclerotic plaques in the aorta, and in nine of the controls, at least 13% of the entire surface was involved. Intimal thickness ranged up to 390 mum. In contrast, four of the pigs with vWd did not develop such lesions, two developed arteriosclerotic lesions affecting 6 and 7% of the aortic surface, and the seventh had 13% of the aortic surface involved. Most of the pigs with vWd, however, developed flat fatty lesions in contrast to the normal pigs whether on the normal or the high cholesterol diet. There was blue staining of the flat fatty lesions when two pigs with vWd were injected with Evans blue dye antemortem. By electron microscopy, severe endothelial damage was apparent, but there was no intimal proliferation. The coincidence of the impaired platelet-arterial wall interaction and lack of arteriosclerosis in this bleeding disease is discussed.

Molecular cloning, functional expression and localization of an inward rectifier potassium channel in the mouse brain

We have cloned a novel inward rectifier potassium channel from a rat brain cDNA library and designated it RB‐IRK2. The rat brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. The amino acid sequence of RB‐IRK2 shares 70%, 40% and 45% identity to mouse IRK1, rat ROMK1 and rat GIRK1, respectively. Xenopus oocytes injected with cRNA derived from RB‐IRK2 expressed a potassium current which showed inward‐rectifying channel characteristics similar to the IRK1 current, but distinct from the ROMK1 or the GIRK1 currents. However, the localization of RB‐IRK2 mRNA in rat tissues, assessed by the Northern blot analysis, differed from that of mouse IRK1. These results indicate that the IRK family is composed of multiple genes, which express in different tissues and therefore may play heterogenous functional roles in various organs, including rat central nervous system.

Increased von Willebrand factor in diabetes mellitus.

Abnormal platelet function may play a role in the genesis of vascular complications in diabetes mellitus. We measured plasma levels of ristocetin-Willebrand factor and factor VIII antigen in 75 subjects and also measured aggregation-enhancing factor in subsets. We found increased levels of ristocetin-Willebrand factor in all groups of diabetics studied, even in mild diabetics free of vascular disease. Factor VIII antigen was increased only in diabetics with vascular disease. We could not find an aggregation-enhancing factor in any group.

The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions

A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.

Factor VIII and factor VIIIa.

Publisher Summary This chapter focuses on the role of factor VIII and factor VIIIa present in blood. The factor VIIIa apparently alters the active site structure of factor IXa to make it recognize the rate-limiting transition state for factor X activation, because its dominant kinetic effect is to increase the kcat. This chapter discusses the structure of factor VIII. Electrophoretic analysis of factor VIII preparations is commonly used to evaluate impurities and to determine whether factor VIII has been unintentionally activated. Assays for factor VIII coagulant function continue to play an important role in the assessment of factor VIII preparations. These assays are based on the ability of factor VIII to shorten the clotting time of plasma derived from a patient with hemophilia A. Two types of assays are commonly employed: the one-stage assay and the two-stage assay. The ability to modify a protein covalently at unique locations with fluorescence reporter groups can provide information on intra- and inter-molecular interactions. Factor VIII possesses no active site so that labeling at a single, unique position is difficult.

Arteriosclerosis in normal and von Willebrand pigs: long-term prospective study and aortic transplantation study.

In a long-term prospective study, five normal control pigs and five pigs with homozygous von Willebrands disease received a nonfatty diet from age 3 months to 4 years; then the aortas were analyzed. The fibrous arteriosclerotic plaques in the distal abdominal aortic region involved an average of 28% of the surface area in control pigs and only 7% of the surface area in pigs with von Willebrands disease (F < 0.01). In a subsequent study of 3-month-old pigs, the distal abdominal aortic segments from nine normal pigs were cross-transplanted with segments from nine other normal pigs (control study), and aortic segments from four normal pigs were transplanted into four host pigs with von Willebrands disease (exchange study). All pigs received a 2% cholesterol diet for up to 6 months; then the transplanted aortic segments were analyzed. The donor normal aortic segments in the host normal pigs developed arteriosclerosis that involved an average of 20% of the surface; the endothelial fluorescent pattern of von Willebrand factor was identified. In contrast, the donor normal aortic segments in the host pigs with von Willebrands disease had arteriosclerosis that involved an average of only 4% of the surface (P < 0.01); the endothelial cell von Willebrand factor was not identified. The long-term prospective study indicates that pigs with von Willebrands disease are resistant to the development of spontaneous arteriosclerosis. The aortic transplantation data are compatible with the hypothesis that the absence of von Willebrand factor in pigs with von Willebrands disease may cause impairment of platelet-arterial wall interaction and resistance to arteriosclerosis.

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegeners granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3‐ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N‐terminal activation dipeptide of PR3 is required for the binding of PR3‐ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N‐terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3‐S176A and δ‐rPR3‐S176A. rPR3‐S176A contains the N‐propetide Ala‐2‐Glu‐1, δ‐rPR3‐S176A does not. Culture supernatants of rPR3‐S176A and δ‐rPR3‐S176A expressing 293 cells were used as sources of target antigen for PR3‐ANCA testing by capture ELISA. Forty unselected consecutive PR3‐ANCA+ sera were tested. With δ‐rPR3‐S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3‐S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N‐terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3‐ANCA. However, a substantial proportion of PR3‐ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N‐terminal processing. In 15% of sera this PR3‐ANCA subset occurred exclusively. PR3‐ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non‐haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA

We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC‐1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N‐methoxysuccinyl‐Ala‐Ala‐Ala‐Pro‐Val‐pNA. The enzymatic activity is inhibited by greater than 95% by α1‐PI. The rPR3 and the enzymatically inactive mutant rPR3‐S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3s targeting to granules. This rPR3 is the first to be recognized by most c‐ANCA from WG patients and all anti‐PR3 ANCA that were detected by standard anti‐PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure‐function relationships and interaction with autoantibodies.

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3‐related autoimmune processes investigated in wild‐type‐, mNE‐ and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico‐chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc‐AAPV‐pNA and Suc‐AAPV‐pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by α1‐antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom‐designed hNE inhibitor, Val15‐aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species‐specific assessment of neutrophil protease function and inhibition.

Warfarin or low-molecular-weight heparin therapy does not prolong pig-to-primate cardiac xenograft function

Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low‐molecular‐weight heparin (LMWH) anti‐coagulation on xenograft function using a heterotopic pig‐to‐primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti‐CD20 and TPC, an α‐galactosyl‐polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti‐coagulant drugs. The median duration of xenograft function was 20 days (range 3–62 days), 18 days (range 5–109 days) and 15 days (range 4–53 days) in Groups 1 to 3 respectively. Anti‐coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti‐factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti‐coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti‐coagulant strategies to achieve long‐term xenograft function cannot be excluded.