Bowie, E. J. Walter

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 When oxalated or resin-decalcified human plasma was adsorbed with barium sulfate, equilibrated with 0.0175M phosphate buffer (pH 6.3), and chromatographed through diethylaminoethyl (DEAE) cellulose, most of the plasma’s fibrinogen emerged from the column and factor XII was detected. No factor VIII was found. A second buffer phase, in which 0.04M phosphate buffer (pH 5.9) was used, removed more protein but not fibrinogen or factor VIII. A third phase, 0.10M phosphate buffer (pH 5.6), produced additional fibrinogen and, unlike the first fibrinogen fraction, factor XIII; but again, no factor VIII. A fourth and final phase, 0.40 to 0.50M phosphate buffer (pH 5.3), removed factors VIII and V, and the eluate contained no fibrinogen. Although separation of fibrinogen into two components required the four-buffer system, maximal yields of factor VIII were achieved by compressing the first three buffer phases into one. Specific activities of about 50-fold, from large volumes of plasma, and 200-fold, from small volumes, were obtained. Refined factor VIII was unstable unless citrate was added to a concentration of 0.4 per cent.

Type IIE von Willebrand disease has previously been identified in a single family with two affected members. We have identified a second family with five affected individuals. The pattern of inheritance is autosomal dominant and the clinical picture is characterized by mild mucocutaneous bleeding. Laboratory results for affected patients: VIII:C ranged from .62 u/ml to 1.0 u/ml, VWD antigen 2.0 u/ml to 4.75 u/ml, and ristocetin cofactor activity 0.04 u/ml to .32 u/ml. Bleeding times, PT and APTT results were within normal ranges.Multimeric analysis of both citrated plasma and platelet lysates utilizing sodium dodecyl sulfate-polyacrylamide electrophoresis was characterized by a decrease of the largest oligomers and the individual multimers were abnormal with a single identifiable band. Plasma samples collected in the presence of a variety of inhibitors had no effect on the multi-meric pattern. Intravenous infusion of DDAVP (.3 ug/kg of body weight) resulted in a slight increase in the highest molecular weight oligomers but no change in the ristocetin cofactor activity or individual multimeric patterns.

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.

Measuring the release of small amounts of a clotting factor from an isolated perfused rat liver is difficult if the perfusate already contains some of the factor. Further, platelet-containing perfusates generate a coagulant activity that may invalidate clotting assays.We have successfully employed a completely synthetic blood substitute for rat liver perfusions. The perfusate is “Fluosol-43” generously furnished by Alpha Therapeutic Corporation. The oxygen-carrying perfluorochemical is FC-43 (perfluorotributylamine) and the substitute for albumin is hydroxyethyl starch. Using the Brauer perfusion technique, we found that rat livers in 5 hours released an average of 2.3% of the normal plasma concentration of prothrombin, 8.4% factor V, 16.2% factor VII, 7.0% factor IX, 3.7% factor X, 28.3% factor XI and 12.3% factor XII. Antithrombin III and plasminogen were also generated.Only minute amounts of factor VIII were released unless serum, cryoprecipitate or cryoprecipitate-free plasma was added; then the yield was 8.8% on average. The more “venom factor” (platelet aggregability with Bothrops alternata venom) added to the synthetic perfusate, the more factor VIII was released.

SummaryIn order to evaluate a possible role played by platelets in the development of atherosclerosis, the aortas of 11 control pigs and 11 homozygous von Willebrand (vWd) pigs were examined for spontaneous atherosclerosis. Of the 11 normal pigs, 6 showed multiple atherosclerotic plaques with an intimal thickening of 63 to 130 μm. In contrast, none of the von Willebrand pigs had multiple plaques and only one showed a single lesion of more than 2 mm in diameter.In a prospective study 5 control pigs and 5 vWd pigs were given a high (2%) cholesterol diet from the age of 3 to 9 months. All of the controls developed atherosclerotic plaques. In 4 of the pigs the plaques exceeded 13% of the aortic surface with an intimal thickening of 50 to 390 pm. In contrast, only one of the vWd pigs developed atherosclerotic plaques which only involved 7% of the aortic surface. Most of the vWd pigs, however, developed non-atherosclerotic flat fatty lesions. These findings may be related to the imp aired platelet arterial wall interaction in vWd. The vWd pigs seem to be an ideal model for atherosclerosis research.