Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function

Abstract

The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

Keywords: Bruton's tyrosine kinase; glycoprotein VI; ibrutinib; platelets.

Fig. 1.

A: washed human platelets (5 × 10⁸/ml) were stimulated with 0–10 μg/ml collagen-related peptide (CRP) and subsequently lysed and blotted for Bruton's tyrosine kinase (Btk), phosphorylated Btk (pBtk), and phosphorylated Syk (pSyk). B: washed human platelets were stimulated with 1 μg/ml CRP for 0–5 min, lysed, and then blotted for Btk and phosphorylated Btk. C: washed human platelets were treated with vehicle (DMSO, 0.1%), BTKI-43607 (5 μM), or BTKI-43761 (5 μM) and stimulated with 1 μg/ml CRP for 5 min. Platelets were subsequently lysed and blotted for phosphotyrosine (pTyr) moieties with 4G10 antiserum, as well as phosphorylated Btk and phosphorylated Lyn (pLyn). The surface marker CD31 served as a loading control. Data are representative of 3–5 experiments. MW, molecular weight; PP2, protein phosphatase 2.

Fig. 2.

Flow cytometry analysis of platelet surface P-selectin expression following treatment with vehicle (DMSO, 0.1%), BTKI-43607, or BTKI-43761 prior to stimulation with CRP (10 μg/ml) or thrombin (Thr, 1 U/ml). MFI, median fluorescence intensity. Values are means ± SE; n = 4. *P < 0.05 vs. vehicle.

Fig. 3.

Replicate samples of washed human platelets (2 × 10⁷/ml) treated for 10 min with vehicle (DMSO, 0.1%), the Src kinase inhibitor PP2 (10 μM), or BTKI-43607 or BTKI-43761 (3 or 10 μM) were allowed to spread on immobilized CRP (50 μg/ml), fibrinogen (FG, 50 μg/ml), or collagen (Coll, 100 μg/ml) at 37°C in the absence or presence of the ADP scavenger apyrase (apy). After 45 min, platelets were fixed and mounted onto slides. A: differential interference contrast microscopy images. Scale bar = 10 μm. B–D: surface area of spread platelets. Values are means ± SE; n = 3. *P < 0.05 vs. vehicle.

Fig. 4.

Whole blood was treated with vehicle (DMSO, 0.1%), BTKI-43607 (1 μM), or BTKI-43761 (1 μM), perfused at 2,200 s⁻¹ through glass capillary tubes coated with collagen (100 μg/ml), and surface-blocked with denatured BSA at 37°C to form platelet aggregates. A: aggregates were visualized with differential interference contrast microscopy. Scale bar = 10 μm. B: percent surface area covered by aggregates was computed by outlining and quantifying platelet aggregates over 3 fields of view for each condition. Values are means ± SE; n = 3–4. *P < 0.05 vs. vehicle.

Fig. 5.

Platelet-rich plasma (PRP) from nonhuman primates was stimulated with CRP (1 μg/ml) and analyzed by Born aggregometry. Representative traces are shown for PRP from nonhuman primates (n = 2) dosed with BTKI-43607 or BTKI-43761 from the short-term study.

Fig. 6.

PRP from nonhuman primates was stimulated with CRP (1 μg/ml) and analyzed by Born aggregometry. Change in optical density was recorded as a vertical drop to quantify the extent of the inhibition of platelet aggregation (see Table 2). Traces are shown for PRP from nonhuman primates A and B dosed with BTKI-43607 or BTKI-43761 from the long-term study.