Certain purified hemoglobins are under consideration as potential blood substitutes. We have studied the effects of such hemoglobins on platelet reactivity in plasma or in buffered salt solutions. Platelet responses to agonists were assessed by aggregation, calcium influx, serotonin release and by changes in platelet membrane proteins, determined in isolated membranes by SDS-PAGE and in intact platelets by flow cytometry. Initial studies indicated that ultra purified hemoglobin Ao and the diaspirin crosslinked hemoglobin DBBF-alpha did not activate platelets when added alone but greatly potentiated platelet responses induced by agonists. This effect of hemoglobins was not uniform among the agonists screened. Strong potentiation was seen with arachidonic acid and collagen; variable potentiation with thrombin and no potentiation with ADP, ADP/epinephrine or thapsigargin. Our work has focussed on the effect of the hemoglobins on arachidonic acid-induced platelet activation. Hemoglobins at concentrations of 10-9 - 10-6 M potentiated submaximal (20%) platelet response induced by low concentration arachidonic acid to reach maximal (100%) levels. This potentiation was dependent on the presence of divalent cations but not on a functional platelet cyclooxygenase. Current studies on potential mechanisms include: 1.) action of hemoglobin through an adrenergic receptor, in a manner analogous to epinephrine, 2.) binding of platelet generated nitric oxide by hemoglobin, thereby blocking an inhibitory NO-mediated pathway and 3.)heme-mediated oxygen radical production. Comparison of hemoglobins with different abilities to generate free radicals or with different nitric oxide binding characteristics will be used to determine which of these mechanisms are involved. The significance of this project lies in understanding of the hemoglobin- platelet interaction which may account for some of the toxicity experienced with the use of purified hemoglobins in vivo.
