Factor V binding to human platelets: the role of divalent cations and of platelet preparatory methods

The binding of bovine factor V to human platelets has been studied to ascertain the influence of divalent cations as well as the manner of preparation of platelet suspensions. The binding of factor V to platelets was inhibited by EDTA and EGTA and required calcium (optimum concentration 5mM) but not magnesium. Factor V bound to a single class of low affinity sites in unstimulated gel filtered platelets with a Kd of 3.42 x 10(-8)M. Approximately, 2,340 factor V molecules were bound per platelet. Stimulation with the calcium ionophore A23187 or ADP and fibrinogen and inhibition with prostaglandins E1 (PGE1) or prostaglandins I2 (PGI2) failed to alter these constants. However, washing platelets by repeated centrifugation even in the presence of inhibitors of platelet activation decreased the number of binding sites to 1200 without change in the kd. These studies demonstrate a requirement for calcium in the physiological range for factor V binding to platelets, establish that washed platelets bind fewer factor V molecules than gel filtered platelets, and indicate that stimulation or inhibition of platelet function does not affect factor V binding.