The Effect Of Antiporcine Willebrand Factor Monoclonal Antibodies On The Bleeding Time

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.