Figure 6
Figure 6. Ap4A and NPP2 fail to elicit platelet aggregation. (A) Light transmission aggregometry demonstrates the lack of effect of Ap4A alone or in conjunction with NPP4 on triggering platelet aggregation. Ap3A (80μM) in the absence of NPP4 (blue curve) triggers a primary wave of aggregation, which is followed by rapid disaggregation, while in the presence of 100nM NPP4 (red curve) strong primary and secondary aggregation waves are observed. Conversely, 80μM Ap4A either in the presence (green curve) or absence (black curve) of 100nM NPP4 does not trigger even a primary wave of aggregation. (B) Ap4A has an inhibitory effect on Ap3A-induced platelet aggregation in the presence of NPP4. NPP4 (100nM) alone (blue curve) or in the presence of 80μM Ap4A (red curve) does not trigger platelet aggregation, while 100nM NPP4 in the presence of 80μM Ap3A triggers platelet aggregation (black curve). Addition of 80μM Ap4A to 100nM NPP4 and 80μM Ap3A results in an intermediate degree of platelet aggregation compared with 100nM NPP4 and 80μM Ap3A alone, suggesting that Ap4A may compete with Ap3A for the active site of NPP4. Ap4A and its product ATP are also known to inhibit the P2Y12 receptor. (C) NPP2 has no effect on platelet aggregation in the presence of Ap3A. Ap3A (80μM) in the absence of NPP2 (blue curve) triggers a primary wave of aggregation, which is followed by rapid disaggregation. Nearly identical responses are observed with the addition of 100nM (black curve) and 200nM (red curve) NPP2, suggesting that NPP2 lacks the ability to hydrolyze Ap3A to form ADP. Strong platelet aggregation is observed in the presence of 80μM Ap3A and 50nM NPP4 (green curve). (D) Light transmission and lumi aggregometry performed simultaneously demonstrate strong platelet aggregation and dense granule release, respectively, when platelets are exposed to 100nM NPP4 and 40μM Ap3A. LTA curves are shown originating from the top. The corresponding lumi aggregometry curves, generated by bioluminescent determination of ATP, which reacts with firefly luciferin and luciferase, are shown on the bottom. Addition of 40μM Ap3A alone (blue curves) to PRP results in a primary wave of aggregation, followed by rapid disaggregation and a corresponding lumi aggregometry curve that has an initial small spike at mixing followed by steady decay. A similar pattern is observed when 100nM inactive T70A mutant of NPP4 is mixed with 40μM Ap3A (red-orange lines). In contrast, mixing 100nM NPP4 with 40μM Ap3A (black lines) results in the enzymatic production of ADP causing a primary wave of platelet aggregation to occur, leading to granule release after ∼1.5 minutes, detected as a strong surge of luminescence corresponding to the ATP liberated from the dense granules. The secondary wave results in a stable aggregate. Mixing 100nM NPP4 with 40μM Ap4A (green lines) looks quite different. Because no ADP is produced, there is no platelet aggregation and no granule release. Instead, the luminescence detects the slow and steady enzymatic production of ATP from the moment of mixing.

Ap4A and NPP2 fail to elicit platelet aggregation. (A) Light transmission aggregometry demonstrates the lack of effect of Ap4A alone or in conjunction with NPP4 on triggering platelet aggregation. Ap3A (80μM) in the absence of NPP4 (blue curve) triggers a primary wave of aggregation, which is followed by rapid disaggregation, while in the presence of 100nM NPP4 (red curve) strong primary and secondary aggregation waves are observed. Conversely, 80μM Ap4A either in the presence (green curve) or absence (black curve) of 100nM NPP4 does not trigger even a primary wave of aggregation. (B) Ap4A has an inhibitory effect on Ap3A-induced platelet aggregation in the presence of NPP4. NPP4 (100nM) alone (blue curve) or in the presence of 80μM Ap4A (red curve) does not trigger platelet aggregation, while 100nM NPP4 in the presence of 80μM Ap3A triggers platelet aggregation (black curve). Addition of 80μM Ap4A to 100nM NPP4 and 80μM Ap3A results in an intermediate degree of platelet aggregation compared with 100nM NPP4 and 80μM Ap3A alone, suggesting that Ap4A may compete with Ap3A for the active site of NPP4. Ap4A and its product ATP are also known to inhibit the P2Y12 receptor. (C) NPP2 has no effect on platelet aggregation in the presence of Ap3A. Ap3A (80μM) in the absence of NPP2 (blue curve) triggers a primary wave of aggregation, which is followed by rapid disaggregation. Nearly identical responses are observed with the addition of 100nM (black curve) and 200nM (red curve) NPP2, suggesting that NPP2 lacks the ability to hydrolyze Ap3A to form ADP. Strong platelet aggregation is observed in the presence of 80μM Ap3A and 50nM NPP4 (green curve). (D) Light transmission and lumi aggregometry performed simultaneously demonstrate strong platelet aggregation and dense granule release, respectively, when platelets are exposed to 100nM NPP4 and 40μM Ap3A. LTA curves are shown originating from the top. The corresponding lumi aggregometry curves, generated by bioluminescent determination of ATP, which reacts with firefly luciferin and luciferase, are shown on the bottom. Addition of 40μM Ap3A alone (blue curves) to PRP results in a primary wave of aggregation, followed by rapid disaggregation and a corresponding lumi aggregometry curve that has an initial small spike at mixing followed by steady decay. A similar pattern is observed when 100nM inactive T70A mutant of NPP4 is mixed with 40μM Ap3A (red-orange lines). In contrast, mixing 100nM NPP4 with 40μM Ap3A (black lines) results in the enzymatic production of ADP causing a primary wave of platelet aggregation to occur, leading to granule release after ∼1.5 minutes, detected as a strong surge of luminescence corresponding to the ATP liberated from the dense granules. The secondary wave results in a stable aggregate. Mixing 100nM NPP4 with 40μM Ap4A (green lines) looks quite different. Because no ADP is produced, there is no platelet aggregation and no granule release. Instead, the luminescence detects the slow and steady enzymatic production of ATP from the moment of mixing.

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