Abstract
Background: Dual antiplatelet therapy (DAPT) with acetylsalicylic acid (ASA, aspirin) and P2Y12 inhibitors has improved outcomes in patients with acute coronary syndrome. However, major bleeding complications have compromised the benefits of these drugs and there is currently no effective therapy for excessive bleeding or antidote before urgent surgery. Room temperature-stored platelet transfusions (RSP) can reverse the effect of ASA, however they are not effective at reversing the effects of P2Y12 inhibitors. Cold-stored platelets (CSP) were the standard of care in the 60s and 70s, but they were abandoned, when reduced survival was noted in radiolabeling studies. CSP may have several advantages over RSP in this setting including a state of pre-activation and therefore a potentially superior hemostatic function. For actively bleeding patients, requiring therapeutic transfusions, CSP may be the better product, but CSP have never been investigated for the reversal of DAPT.
Methods/Results: Platelet responses to arachidonic acid and ADP, which are critical for the pathways inhibited by ASA and clopidogrel, were tested. We found that αIIbβ3 integrin activation, measured by the activation specific PAC-1 antibody by flow cytometry, is significantly better in platelets stored up to 15 days in the cold compared to platelets stored for 5 day at room temperature. Even 15 day cold-stored platelets treated with arachidonic acid gave a similar response to fresh platelets. Similarly, in dose response experiments using standard light transmission aggregometry we found significantly superior responses to arachidonic acid and ADP over a wide range of doses with 5 day CSP compared to 5 day RSP. The CSP response to arachidonic acid was very similar to freshly prepared platelets from low to high doses.
To test in vitro reversal, we treated platelets with the in vitro P2Y12 inhibitor 2-MeSAMP which resulted in significantly reduced aggregation in response to ADP. When platelets stored for 5 days in the cold were added one minute prior to stimulation with 10 µM ADP, the aggregation response was similar to that observed after adding freshly isolated platelets and significantly better than with platelets stored for 5 days at room temperature.
To test whether CSP are better than RSP in promoting adhesion and aggregation under flow conditions we performed flow chamber experiments. Chambers were coated with 400µg/ml collagen and perfused with arterial shear (1500 1/s) for 5min. We treated C57BL/6J mice with a loading dose of DAPT and labeled, DAPT inhibited, native mouse platelets with a commercially available, fluorescently labeled GPIb antibody ex vivo. Calcein-AM labeled human RSP or CSP were added to the labeled mouse whole blood and subsequently perfused. We found the mean fluorescence intensity to be significantly higher with CSP compared to RSP over time, highlighting a superior reversal effect under flow conditions.
To test CSP and RSP for DAPT reversal in a mouse model, we treated NOD/SCID mice with a loading dose of DAPT. We cut 2mm of the distal tail and collected blood over 30 minutes, followed by hemoglobin concentration measurement in the collected fluid. Interestingly, retroorbital transfusion of human 5 day stored RSP, resulted in significantly more blood loss compared to the group without transfusion. CSP-transfused mice lost less blood than the RSP-transfused mice, but no significant difference was observed when CSP-transfused mice were compared to the group without transfusion.
Conclusion: Our in vitro and in vivo data indicate that CSP could have a critical advantage over RSP when reversal of DAPT is required.
Disclosures
No relevant conflicts of interest to declare.
Remote Ischaemic Preconditioning Causes Pathway Specific Attenuation of Platelet Glycoprotein IIb-IIIa Activation in Response to Potent In-Vitro Agonist Stimulation in Spite of Dual Antiplatelet Therapy
