RIPA was investigated using purified human factor VIII (F VIII) and formalin-treated washed platelets suspended in tris buffered saline. At constant (F VIII), aggregation was very sensitive to the conc, of Ristocetin (R) in the range 0.2-0.5 mg/ml, but higher (R) did not further increase aggregation. At constant (R), increasing (F VIII) caused increased aggregation for any (F VIII) used (0.3-3.0 μg protein/ml, 0.015-0.15 U F Vlll-related ant ig en/ml).RIPA was inhibited by low conc. (0.15 mg/ml) of the gelatin plasma expander Haemaccel (H) (Behringwerke). Addition of H after aggregation was complete caused reversal of the aggregation. The inhibition could be overcome by increasing (R), but not by increasing (F VIII) in the conc, ranges used. The conc, of R which overcame H inhibition also caused precipitation of H. This precipitation could be reversed by increasing (H). Our evidence suggests that H inhibits RIPA by binding R, thus inhibiting the interaction of R and F VIII on the platelet surface which is responsible for aggregation.Human Fibrinogen (Fb) (Kabi, 99% pure, 0.5 U F Vlll-related antigen/mg) caused a similar inhibition of RIPA in conc, as low as 0.2 mg/ml. Higher conc caused greater inhibition, which could be overcome by increasing (R). The Fb (2.5 mg/ml) itself did not induce aggregation in the presence of R. R may also bind to other plasma proteins as well since it can form a precipitate with serum.These findings are of considerable importance for the quantitative measurement of the Ristocetin co-factor. RIPA is very sensitive to a small range of (R) and the availability of R in the test system can be greatly affected by the presence of Fb and other plasma proteins.
Support of ristocetin-induced platelet aggregation by procoagulant- inactive and plasmin-cleaved forms of human factor VIII/von Willebrand factor
