Fass, David N.

SUMMARY Normative values were established for results of tests of blood coagulation and platelet function in swine. In swine that have von Willebrand's disease, bleeding time was prolonged, platelet retention was decreased glass bead column), and factor VIII concentration was low.

SUMMARY Factor VIII concentrations in plasma, platelet retention, and ear bleeding times were determined on 35 normal pigs, 40 obligate carriers of porcine von Willebrand's disease, and 25 bleeders. The bleeders could be distinguished from the normal pigs by each of the 3 tests. At most, only one-third of the carriers could be distinguished from the normal pigs by any one test. A hemostatic score, derived from the results of the 3 tests, increased the discrimination to slightly more than half. The degrees of abnormality on the 3 diagnostic tests seemed to vary from family to family and might have been related to the sire of the bleeder.

Abstract Factor VIII (FVIII) inhibitors have been classified as Type I, linear inactivation kinetics and complete inhibition of FVIII at saturation, or Type II, non-linear kinetics and residual FVIII activity. To investigate the hypothesis that "inhibitor-blocking antibodies" contribute to the characteristics of Type II inhibitors, mixing studies were performed between fractionated Type II antiserum and various Type I inhibitor preparations. Type II IgG fractions were tested for inhibitory capacity and ability to prevent inhibition mediated by inhibitors from other hemophiliacs. The IgG from Type II plasma (II) (29 BU, 8% FVIII) and several Type I plasmas ((I) plasmas) were fractionated with DEAE resin. Late-eluting Type II fractions (II):6 and (II):7, showed slight protection of FVIII from the inhibiting activity of several Type I plasmas and additive inhibition with others. When tested against Type I IgG fractions instead of type I plasmas, (II):6 and (II):7 provided significant protection of FVIII from the inhibitory action of some immunoglobulins and additive inhibition with others. When (II):6, which contained slightly less than 1 BU of inhibitory activity, was preincubated with normal plasma, it blocked about 3 BU of inhibitory activity in an equal volume of fraction (I):1; residual FVIII increased from 11.4% to 27.2%. IgG fraction (II):6 provided protection from DEAE fraction 2, (I):2, from another type I patient (residual FVIII went from 15.1% to 31.7%) but was additive with (I):4 from the same patient (residual FVIII went from 8.0% to 3.2%). Preincubation with (II):7 increased residual FVIII activity in normal plasma incubated with (I):1 from 10.1% to 20.7%. Fraction (II):7 was processed further by hydrophobic interaction chromatography on phenyl sepharose and two fractions, Phen(II):5 and Phen(II):6 provided significant protection of FVIII from inhibition by (I):1; residual FVIII activity increased from 11.5% to 34.1% and from 7.9% to 28.2%, respectively. DEAE fractions (II):6 and (II):7 were shown to contain anti-FVIII IgG. Depletion of mixtures of plasma or rFVIII and (II):6 with immobilized Protein A, reduced FVIII activity by 42.8% and 68.8%, respectively. In addition, adsorption of (II):6 with immobilized Protein A, eliminated its ability to block FVIII inhibition by (I):1. The co-existence of these antibodies in "acquired hemophiliac" plasma would contribute to the complex kinetics seen in these patients. If a sufficiently diverse panel of such blocking IgGs occurred naturally, or could be produced in the laboratory, and their interaction with the target antigen did not adversely affect the half-life, then these blocking antibodies might prove useful to alleviate the effects of inhibitory antibodies in hemophilia and other diseases.

When isolated neonatal pig livers were perfused with blood from pigs with von Willebrand’s disease, no factor VIII coagulant activity (VIII:C) emerged in the perfusate. When purified porcine Willebrand factor, lacking VIII:C, was added to the perfusate it rapidly diminished, presumably being taken up by the liver. Concurrently VIII:C rose steadily in the blood during the 5 hours of perfusion. Similarly, isolated adult rat livers released no VIII:C when they were perfused with rat erythrocytes suspended in Tyrode’s solution containing 6% bovine serum albumin. VIII:C did emerge if cryoprecipitate from aged rat plasma were added. The neo-VIII:C was rapidly inactivated by plasma from a patient with classic hemophilia who had developed an antibody to VIII:C Addition of cycloheximide to the rat before extirpation of the liver, as well as to the perfusate, prevented the emergence of VIII:C. We suspect that VIII:C is derived from the liver but only when Willebrand factor is present.

Platelet-arterial wall interaction appears to be important in the genesis of atherosclerosis. Pigs with homozygous von Willebrand’s disease (vWd) appear resistant to atherosclerosis. We investigated whether there is impairment in platelet-arterial wall interaction in porcine vWd. Superficial endothelial injury was produced in a 4 cm segment of carotid artery by drying for 3 minutes in a stream of air which entered one end (100 ml/min) and exited through a small hole in the other end. This procedure was performed in 9 normal and 6 vWd pigs 24 hours after the I.V. infusion of III-Indium-labeled platelets. Carotid blood flow was re-established and the pigs were sacrificed 24 hours later and the carotids fixed in glutaraldehyde.The ratio of radioactivity (cpm/g) of injured/noninjured carotid arteries was markedly decreased in the vWd pigs. In addition, transmission and scanning electron microscopy confirmed superficial endothelial damage in all injured segments, but only significant platelet deposition in the normal pig injured segments.After a mild endothelial injury, this “in vivo” evidence of impaired platelet-arterial wall interaction in vWd may be related to the resistance to atherosclerosis in these animals.

Several laboratories report factor VIII coagulant (VIII: C) from human, bovine and porcine sources interact with Willebrand factor (WF). Bovine coagulant may be S-S bonded to WF: the human and porcine VIII:C may reversibly bind to WF. This interaction has been used by various laboratories to help purify the VIII:C. The VIII:C from all 3 species are activated by Ila and inactivated in EDTA. Our work has involved the porcine proteins bound by WF and anti-VIII:C antibodies. Human antibodies to human VIII:C bind porcine plasma proteins which on SDS-PAGE band at apparent MW 160K, 130K and 80K. These data are consistent with the human VIII:C (Switzer and McKee (JCI 60:819, 1977)). Their human material appears to contain S-S linked chains, porcine chains separate in SDS alone. Thrombin treatment results in loss of high molecular weight proteins and an appearance of activation/deactivation products at MW = 80K and 73K and lower. Two murine monoclonal anti-porcine VIII:C antibodies partially inactivate VIII:C while a third doesn’t. This latter antibody, when immobilized, removes activity from solutions of VIII:C dissociated from WF but not from solutions of undissociated VIII:C. The VIII:C immobilized through this antibody is still active, can be removed by organic solvent in active form, and contains the same protein chains as those bound to the polyclonal human anti- VIII:C. The monoclonal antibody VIII:C complex is inactivated and releases the 130K + 160K chains in EDTA. The 80K MW chain can be removed separately. Based on relative staining intensity in SDS gels, the porcine VIII:C at this stage of purity may be heterogeneous, (130K + 80K and 160K + 80K), with the chains associated through Ca++. The 80K MW chain, may bind the VIII:C to porcine WF. The site of this interaction does not seem to be critical to the measurement of VIII:C activity in vitro.

Murine monoclonal antibodies to porcine Willebrand factor were used to study the role of Willebrand factor in hemostatic plug formation in the in vivo and in vitro skin bleeding times. The in vivo assay requires the intravenous injection of antibody-containing mouse ascitic fluids into normal animals with subsequent ear bleeding time tests performed over a 24-hour period. The in vitro assay was carried out with heparinized normal porcine blood flowing through a 0.5 cm incision in an excised piece of porcine skin. The heat inactivated ascitic fluids, containing the antibodies, were used ;Ln these assays at final dilutions of 2 × 102 through 2 × 105 The 7 antibodies used comprised at least 5 groups with differing reactivities based on assays other than bleeding time tests. The antibody titers in a poreineg Willebrand factor-binding radioimmunoassay ranged from 108 through 1012 . Doses of 10HL of ascitic fluid/kg of body weight (approximately 2 × 103 final dilution) were able to transiently prolong the in vivo bleeding time without alteration of other VIII complex values (VIII:C, ristocetin cofactor activity and Vlll-related antigen). Higher doses resulted in bleeding times similar to von Willebrand pigs (>15 min) immediately following infusion, with a decay of the effect over the next 2 hours. At dilutions of 2 × 104 selected monoclonal antibodies interfered with hemostasis in the in vitro model whereas other antibodies inhibited only at 2 × 102 dilution. With one exception, the potencies of the antibodies appeared to be similar in both assays. The titer of the antibodies in the radioimmunoassay does not appear to correlate with prolongation of the in vitro bleeding time, suggesting specific reactivity with functional sites involved in hemostatic plug formatio.

Abstract Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) ’in vitro' bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R: AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin (’in vitro' bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atheroscolerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.

SUMMARYANCA directed against PR3 are highly specific for Wegener's 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.