Effects of Bovine Factor VIII-Related Protein on Human Platelets and Isolated Human Platelet Membranes

The first phase of aggregation of human platelets induced by bovine factor VIII-related protein does not require an intact energy metabolism and probably represents a passive agglutination phenomenon due to receptors for the protein on the outside of the platelet cytoplasmic membrane. However, washed human platelets lost their ability to be aggregated by bovine factor VIH-related protein after freezing and thawing of the platelets. Furthermore, isolated human platelet membranes were not flocculated by the bovine protein in the absence of added calcium ions. Additions of calcium chloride (2.1 mM) alone flocculated the isolated membranes. The membranes used represented the material sedimenting between 10,000 and 100,00 g from a homogenate obtained by homogenizing washed platelets suspended in 0.27 M sucrose in an Aminco-French pressure cell (1.361 atm. 2 × 1 min). Subsequent sucrose density gradient centrifugation of the preparations did not reveal bands of particulate matter at a higher sucrose density than 1.12. In contrast, freezing and thawing of formalin-treated human platelets (4 mg/ml formaldehyde) did not destroy their ability to be aggregated by bovine factor Vlll-related protein. Application of the method described above to the formalin-treated platelets resulted in a low yield of 10,000-100,000 g sedimenting material and to more particulate bands on subsequent sucrose density gradient centrifugation. The stabilizing effect of the formalin treatment (through protein polymerization ?) may also be seen from the fact that such platelets are aggregated by bovine factor VHI-related protein even after their ability to aggregate with thrombin has been lost.