P2Y₁₂-Independent Transient Activation and P2Y₁₂-Dependent Prolonged Activation of Platelet Integrin α_IIbβ₃.

Affinity of platelet integrin α_IIbβ₃ for ligands is dynamically regulated and the activation of α_IIbβ₃ has been analyzed by binding assays using fibrinogen or ligand mimetic antibodies such as PAC1. Ligand binding assays are suitable for the condition when the number of receptors would reach a plateau phase before the end of incubation period. However, these assays would not give us useful information when the receptors are exposed only transiently followed by rapid inactivation within a few minutes. We found transient aggregation of platelets from a P2Y₁₂-deficient patient (OSP-1) when platelets were stimulated with PAR1-activating peptide (PAR1-AP), PAR4-AP or U46619. Therefore, we hypothesized that transient α_IIbβ₃ activation occurs under P2Y₁₂-deficient conditions. To estimate time-dependent changes of activated α_IIbβ₃, we analyzed PAC1 binding rates by measuring PAC1 binding every 30 sec (or 1 min) after the addition of PAC1 with different post-stimulation lag periods because the initial velocity of ligand binding is known to correlate with the number of activated receptors (Frojmovic et al., Biophys J, 59, 1991). When control platelets were stimulated with PAR1-AP (50μM), PAC1-binding rates did not change up to 5 min. In P2Y₁₂-deficient platelets, the binding rate in the initial 30 sec was equivalent to control platelets and estimated values of activated α_IIbβ₃ in the first time fraction was, at least, about 50 % of PMA-treated platelets regardless of P2Y₁₂-signals. In sharp contrast, only a subtle increase of PAC1 binding was seen in the sixth time fraction (2.5–3min), indicating that majority of activated α_IIbβ₃ was rapidly inactivated within 3 min in P2Y₁₂-deficient platelets. We also found clear, but transient PAC1 binding induced by PAR1 stimulation in megakaryocytic cell line, CMK. When FITC-PAC1 binding was analyzed in CMK expressing high levels of glycoprotein Ib, substantial PAC1 binding was observed 0.5–10 min after PAR1 stimulation. Similar transient PAC1 binding profiles were also observed in primary megakaryocytes derived from cultured umbilical cord blood mononuclear cells. Initial velocities of PAC1 binding with different lag periods confirmed the highest levels of α_IIbβ₃ activation in the first time fraction (0 to 1 min) both in CMK and in megakaryocytes. Next, we found that P2Y₁₂-dependent signals are defective in CMK because intracellular cyclic AMP levels did not change by ADP in PGE1-treated CMK. Overexpression of P2Y₁₂ by nucleofection, however, resulted in ADP-dependent increase of PAC1 binding by PAR1 stimulation and the extent of ADP-dependent effects correlated with the expression levels of P2Y₁₂ both in CMK and in megakaryocytes.

It has been believed that agonist-induced α_IIbβ₃ activation is absent under P2Y₁₂-defective conditions or in megakaryocytic cell line because bound ligand is not observed in “usual” ligand binding assays. However, analyzing PAC1 binding rates in our experimental systems unravels P2Y₁₂-independent transient α_IIbβ₃ activation. Furthermore, we demonstrated that the duration of α_IIbβ₃ activation is prolonged by P2Y₁₂-signals in platelets, CMK and megakaryocytes. Here, this new strategy would let us overcome serious problems existing in “usual” ligand binding assays, providing precise information about dynamic behaviors of integrin α_IIbβ₃.