Program: Oral and Poster Abstracts
Session: 301. Platelet Activation and Biochemistry: Poster II
Methods: To investigate the possibility that bleeding events in Ibrutinib-treated patients are due to inflammatory hemorrhage, we used two inflammatory models - the dermal reverse passive Arthus reaction (rpA) and P. aeruginosa LPS-induced lung inflammation, both of which have been shown to induce hemorrhage in the absence of platelet ITAM signaling. To selectively inhibit Btk in platelets, thrombocytopenic mice were reconstituted with Ibrutinib- or vehicle-treated wild-type platelets and challenged in rpA or LPS model. Bleeding in the dermis or broncho-alveolar lavage (BAL) fluid was assessed by analysis of hemoglobin. In the clinic, patients generally receive once-a-day doses of 420-560 mg of the Btk inhibitor Ibrutinib, and plasma concentrations can transiently reach 0.5-1 µM. Thus, during their lifetime, circulating platelets are exposed to compounding doses of Ibrutinib, which may have greater inhibitory effects than a single dose, a phenomenon which we have observed in mice (see results below). Therefore, we treated mouse platelets in vitro with 0.5 µM Ibrutinib (low dose, equal to a single dose) or 5 µM Ibrutinib (high dose, comparable to multiple dose exposure) before adoptive transfer into mice.
Results: While low dose Ibrutinib did not prevent αIIbβ3 integrin activation induced by collagen or convulxin (Cvx), high dose Ibrutinib abolished collagen-induced aggregation. This dose-dependent inhibition of aggregation was also observed in platelets isolated from mice which had received a single vs. multiple doses of Ibrutinib in vivo. αIIbβ3 activation induced by the stronger GPVI agonist, Cvx, however was only partially impaired, suggesting that Ibrutinib may not completely inhibit ITAM-dependent platelet activation. We next tested whether platelets treated ex vivo with Ibrutinib and transfused into thrombocytopenic mice were able to secure vascular integrity at sites of inflammation. In both models, platelet depletion without transfusion of wild-type platelets induced substantial bleeding. However, transfusion of Ibrutinib-treated platelets was able to rescue bleeding to the same extent as vehicle-treated platelets.
Summary: Our findings demonstrate that Ibrutinib-treated platelets are capable of securing vascular integrity at sites of inflammation in mice. Our studies suggest that bleeding events in patients treated with Ibrutinib are not due to inflammation-associated hemorrhage.
Disclosures: Bergmeier: AHA: Research Funding ; NIH: Research Funding .
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