Biomedical subjects
Publications and source records attributed to D N Fass.
Two Puerto Rican families were studied. One family included a number of members with dysfibrinogenemia occasionally associated with hypofibrinogenemia. The second family had members with von Willebrand's disease. The two diseases merged in the proband's immediate family; the affected members of this family exhibited a mild bleeding disorder. Others in the two families had no obvious bleeding tendency.
Heparinized porcine blood and plasma, at constant hydrostatic pressure, was allowed to flow through a 5-mm incision in a small piece of porcine skin. Changes in the exuded blood volume were measured, and the incision site was examined microscopically. When normal blood flowed through either normal or von Willebrand skin, the exuded blood volume decreased gradually and eventually stopped. Microscopic examination revealed a platelet plug in the incision site. This plug was positive for Willebrand factor when examined by immunofluorescence. In contrast, the blood from von Willebrand pigs continued to flow constantly, and a platelet plug was not seen. The delayed in vitro hemostasis in von Willebrand blood was corrected to the normal range by the addition of either normal plasma or partially purified Willebrand factor. Normal blood, in which the Willebrand factor was immunologically inhibited, showed delayed hemostasis. For this in vitro system, it appeared that plasmatic Willebrand factor played an essential role in hemostasis.
The aortas of 11 pigs (aged 1-3 yr) with homozygous von Willebrand's 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.
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).
Aortic endothelial cells from normal pigs and pigs with von Willebrand disease have been established in long-term cultures. Both cultures appeared similar in terms of general growth characteristics, morphologic features and ultrastructure. Immunofluorescent staining of these cultures with chicken (or rabbit) antiporcine ristocetin-Willebrand factor sera (or IgG) resulted in extensive perinuclear staining of the cells in both cultures. Additionally, staining of semiconfluent cultures of normal cells for ristocetin-Willebrand factor revealed an extensive meshwork of distinct, immunologically identifiable ristocetin-Willebrand factor-containing filaments between cells. Immunoreactive material was considerably decreased and more diffuse between cells in semiconfluent cultures from affected pigs. Through immunocytochemical staining with peroxidase-coupled antiserum, the filaments (of indeterminate length) were found to have a diameter of approximately 300 A. Finally, washed porcine platelets interacted extensively with scrape-damaged cultures of affected endothelial cells. This interaction of platelets with damaged normal cultures was abolished by pretreatment of the cultures with rabbit antiporcine ristocetin-Willebrand factor IgG.
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A plasmatic component required for the ristocetin-induced aggregation of platelets has been purified from normal human and porcine plasma by gel filtration (4% agarose) and anion-exchange chromatography (DEAE cellulose). No factor VIII coagulant activity was found associated with the purified human or porcine component. Urea sodium dodecylsulfate electrophoretic analysis of the purified component of both species indicated that the apparent molecular weight with intact disulfides is in excess of 500,000; after disulfide reduction with 2-mercaptoethanol, single components with an apparent subunit molecular weight of 230,000 were observed. Purified porcine ristocetin-Willebrand factor (RWF) co-sedimented in sucrose gradients with the factor present in normal plasma. Amino acid analysis of both human and porcine RWF indicated that all normal amino acids are present, whereas amino sugars were undetected. However, lipid analysis indicated 1% to 2% lipids present, including monoglycerides, di- and tri-glycerides, cholesterol, cholesterol esters, some free fatty acids, and a trace of phospholipid. A single line of identity was observed between normal human plasma and purified human RWF when immunodiffusion plates were run with purified rabbit anti-human RWF immunoglobulins. Antisera raised against human and porcine RWF's do not inhibit the factor VIII coagulant activity of the homologous plasma, nor is "spontaneously occurring" human factor VIII inhibitor neutralized by the isolated material of either species.
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We have examined nine patients with presumed von Willebrand's disease who present the spectrum of that disorder. Two had findings that would be accepted generally as diagnostic of von Willebrand's disease, and seven had variations of the usual pattern. The commonest variation was the combination of borderline and variable levels of coagulant Factor VIII, commensurate levels of Factor VIII-related antigen, and low levels of ristocetin-Willebrand factor.
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Normal subjects, patients with various bleeding disorders, and patients with von Willebrand's disease were studied. All patients with von Willebrand's disease had decreased levels of ristocetin-Willebrand factor (range, 0 to 41%) as compared with all other subjects (range, 79 to 202%). Ristocetin-induced platelet aggregation of platelet-rich plasma was abnormal in all patients with von Willebrand's disease tested, and it was possible to correct this abnormal response by addition of normal platelet-poor plasma. Abnormal ristocetin-induced platelet aggregation was seen in patients with intrinsic platelet disorders or, on some occasions, in normal patients who had ingested aspirin. Ristocetin-induced platelet aggregation is not diagnostic, but it may be useful as a simple screening test for patients with possible von Willebrand's disease. In conjunction with other tests, the assay for ristocetin-Willebrand factor will be useful in diagnosis and evaluation of these patients.
Nine pigs with von Willebrand's disease (vWd pigs) received normal auxiliary livers; in the immediate postoperative period, three pigs died, three survived from 3 days to 3 weeks, and three were killed 5-13 weeks postoperatively. There was an increase in factor VIII (up to 90 u/dl) but with a type of factor VIII that was more labile than normal and that decreased after 2 weeks, probably as a result of liver rejection. There was a slight increase in the levels of the ristocetin-Willebrand factor postoperatively (up to 11%) but this activity was undetectable after 2 weeks. There was no correction of the abnormal bleeding time and platelet retention. When a vWd liver was transplanted orthotopically into a normal pig, the factor-VIII coagulant activity and the ristocetin-Willebrand factor remained normal. The abnormality in porcine von Willebrand's disease is only partially corrected by liver transplantation, which suggests that there also is extrahepatic synthesis of the missing or abnormal plasmatic activity or activities.
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