Hemoglobin A_oand α-Crosslinked Hemoglobin (α-DBBF) Potentiate Agonist-Induced Platelet Aggregation Through the Platelet Thromboxane Receptor

Abstract

Chemically modified hemoglobins are potential oxygen-carrying blood substitutes, but their in vivo administration has been associated with a variety of unexpected side events, including increased platelet reactivity. We studied the effects of hemoglobin A0 (HbA0) and alpha-crosslinked hemoglobin (alpha-DBBF) on platelets in vitro. Neither hemoglobin A0 nor alpha-DBBF activated platelets when added alone, but both proteins potentiated submaximal agonist-induced platelet aggregation without increasing other markers of platelet activation such as serotonin secretion. Only agonists that are known to cause release of platelet arachidonic acid (AA) were potentiated while aggregation induced by ADP, which does not release AA, was not potentiated. Blockade of the thromboxane receptor with SQ-29,548 prevented the HbA0-induced and the alpha-DBBF-induced potentiation suggesting that the AA/thromboxane signaling pathway mediates the interaction of platelets with hemoglobin.

Hemoglobin-based oxygen carriers do not alter platelet functions: study of three chemically modified hemoglobin solutions

“…This shows that HBOCs does not induce any activation of platelets in vitro. These data were in agreement with those of Mondoro et al [9] who found in vitro no modification in fibrinogen binding by αα-Hb and cell-free Hb in PRP. Indeed, GPIIb/IIIa, the glycoprotein binding to fibrinogen, was not overexpressed in the presence of Hb solutions in our study.…”

“…This finding confirms, in whole human blood, those previously obtained by several authors using other methods. For instance, Pasqui et al [29] and Mondoro et al [9], using a turbidimetric method of aggregometry on human PRP, and Phillips et al [11], working on rat whole blood by impedance aggregometry, have shown that the presence of cell-free Hb does not initiate platelet aggregation. However, our findings differ from those of Iuliano et al [7] who have suggested that the presence of low concentration of cell-free Hb with platelet primed with different agonists should induce platelet aggregation in vitro by generating oxygen free radicals.…”

“…Aggregation patterns were recorded for 15 min after addition of one of the following agonist: collagen (Collagenreagent Horm, Nycomed, Munich, Germany) at the final concentration of 0.5 µg/ml or TRAP (Novabiochem) at the final concentration of 12.5 µM. According to the findings of Mondoro et al [9], platelet aggregation is potentiated by hemoglobin after stimulation by thrombin, collagen, and thromboxane A 2 /prostaglandin H 2 receptor agonist U46619, whereas neither ADP-induced nor ADP together with epinephrine-induced aggregation was affected. Hence we chose to use collagen and TRAP.…”

“…Therefore understanding the impact of HBOCs on platelets becomes essential since some of these solutions are undergoing phase 3 clinical trials in trauma patients and in orthopedic and cardiac surgeries. Moreover, the results of studies addressing the effect of cell-free Hb on platelet aggregation both in vitro [8,9] and in vivo [10,11] are contradictory. Indeed, some authors find a potentiation [9], while others working with the same hemoglobin solution do not [8,11].…”

αα–Crosslinked Hemoglobin: Was Failure Predicted by Preclinical Testing?

“…Eaton [134]expressed concern that hemoglobin products may interfere with normal metabolic mechanisms in the monomacrophage system, amplifying output of proinflammatory cytokines, such as tumor necrosis factor. In fact, both unmodified hemoglobin and DCLHb were found to potentiate submaximal agonist–induced platelet aggregation, effects that could be blocked by administration of a thromboxane receptor blocker [135]. …”