Binding of Factor VIII to Platelets in the Presence of Ristocetin

Abstract

Ristocetin agglutinates platelets in platelet‐rich plasma (PRP) containing factor VIII‐related von Willebrand factor (WF). When the clumps were separated from the supernatant and resuspended in buffer, the platelets dispersed if the buffer was free of ristocetin and the PRP had been treated with aspirin or EDTA to prevent the platelet release reaction. Binding to platelets was determined by measuring WF (ristocetin cofactor assay), VIIIR‐antigen (AG, by radioimmunoassay), and coagulant activity (C) in the initial plasma, the supernatant remaining after removing the clumps formed by ristocetin, and the eluate produced after resuspending the clumped platelets in buffer. No activity was bound to platelets in the absence of ristocetin. After agglutinating platelets in PRP with ristocetin, WF, AG and C activities in the supernatants were about 65% of the initial plasma values, and in the eluates about 9%. Formalin‐fixed washed platelets resuspended in 1 in 3 plasma, and platelets in PRP diluted 1 in 3 also agglutinated when shaken with ristocetin. There was no difference between results with fixed and unfixed platelets. In one series of experiments, the supernatants had about 60% of the initial WF and AG activities; since these activities were the same, they may measure the same substance. In a later series, WF and C were compared; in shaken samples the supernatants had 39% of the initial WF activity and 56% of the initial C activity, and in unshaken samples the supernatants had 3% WF and 29% C. The significantly lower values for C binding suggest that this activity is located on a different molecule from WF‐AG and that the molecules are more readily separated in diluted than in undiluted plasma. Activity in the eluates was inversely proportional to that in the supernatants although the initial activity was never completely recovered. WF and C binding increased with platelet number and ristocetin concentration. Platelets exposed to ADP before fixation showed less agglutination than control fixed platelets and bound less WF. Fixed platelets incubated for 15 min with 30 μg trypsin/ml and washed, or platelets from a patient with the Bernard‐Soulier syndrome failed to agglutinate and bound virtually no WF, AG or C. We conclude that WF, AG and C reversibly bind to fixed or unfixed platelets in the presence of ristocetin, that binding is similar in undiluted plasma but greater for WF and AG than C in plasma diluted 1 in 3, and that ADP and trypsin alter binding, probably by affecting a platelet receptor.