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.

bruton's tyrosine kinase (Btk) is a member of the Tec family of nonreceptor tyrosine kinases that is involved in signaling downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors in hematopoietic cells, including B cells, monocytes, neutrophils, natural killer cells, and platelets (7, 18, 22). Btk was identified in 1993 as the cause of X-linked agammaglobulinemia (XLA), an immunodeficiency disease in which mutations in Btk are associated with a lack of B-cell maturation and, consequently, a low level (<2%) of circulating B cells (18). Patients with XLA show impaired platelet aggregation in response to both collagen and collagen-related peptide (CRP), a membrane glycoprotein (GP) VI (GPVI) agonist, along with decreased activation of the downstream effector protein phospholipase Cγ2 (PLCγ2), suggesting a role for Btk in signaling in platelets (21, 26).

Blood platelets are rapidly recruited to exposed extracellular matrix proteins such as collagen at sites of vascular injury. Initial platelet recruitment to the injury site occurs after circulating von Willebrand factor (vWF) undergoes a conformational change upon binding exposed collagen, followed by platelet receptor GPIb binding to vWF and platelet integrin α₂β₁ binding to collagen (20). Additional platelet adhesion to collagen and subsequent platelet activation are mediated by the platelet receptor GPVI, which is noncovalently associated with a disulfide-linked homodimer of Fc receptor γ-chains, each of which contains an ITAM unit (26). Upon binding exposed collagen, GPVI forms cross-links that enable the GPVI-bound Src kinases Fyn and Lyn to phosphorylate two tyrosines on the Fc receptor γ ITAMs (23). The tyrosine kinase Syk then binds the phosphorylated ITAMs, where it undergoes phosphorylation by the Src kinases and autophosphorylation (26). Activation of Syk initiates assembly and activation of a signalosome, including the transmembrane adapter protein LAT, the cytosolic adapter proteins SLP-76 and Gads, and the Tec kinases Btk and Tec in complex with the effector protein PLCγ2 (26). From this signaling complex, Btk is phosphorylated by Syk and Lyn and autophosphorylated and proceeds to phosphorylate PLCγ2, which hydrolyzes phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing platelet activation through release of intracellular Ca²⁺ stores, activation of protein kinase C, synthesis of thromboxane A₂, and subsequent platelet granule secretion (16, 26).

Several other pathways of platelet activation converge on PLCγ2 and, thus, may require Btk for activation. Outside-in signaling of the fibrinogen receptor integrin α_IIbβ₃ upon binding fibrinogen leads to activation of the Gα protein G₁₃, which initiates c-Src activation of Syk, which, in turn, activates phosphatidylinositol 3-kinase and Btk to phosphorylate PLCγ2 (16). Binding of agonists such as thrombin and thromboxane A₂ to G protein-coupled receptors that activate G₁₃ also initiates this signaling pathway (16). Additionally, shear-dependent binding of the platelet membrane complex GPIb-GPIX-GPV to vWF recruits Lyn to activate phosphatidylinositol 3-kinase, again leading to Btk and PLCγ2 activation (17). Several of these pathways are interdependent and self-amplifying to promote platelet aggregation and formation of a stable thrombus.

In support of its role in B-cell receptor signaling, Btk has been found to be elevated in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and after several successful clinical trials, the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of CLL and MCL (1, 6, 8, 10, 11, 13, 25). A mild bleeding diathesis has been reported in up to 50% of patients taking ibrutinib, with grade 3 or higher bleeding events occurring in up to 6% of these patients (1, 10, 25). Several recent studies suggest that these antihemostatic events may be due to the inhibitory effect of ibrutinib on platelet function (14, 15). To further define the role of Btk inhibition on platelet function, we used two ibrutinib analogs to investigate the effect of irreversible Btk inhibition on platelet activation, spreading, and aggregation.

MATERIALS AND METHODS

List of reagents.

Collagen was obtained from Chrono-Log (Havertown, PA), CRP from R. Farndale (Cambridge University, Cambridge, UK), human fibrinogen from Enzyme Research (South Bend, IN), FITC-conjugated annexin V from Life Technologies, and CD62E-FITC antibody from Acris Antibodies. Antibodies for Western blot experiments were obtained from Santa Cruz Biotechnology (pLyn), Cell Signaling Technology (pBtk and pSyk), and Millipore (4G10). Bovine thrombin, fatty acid-free BSA, and all other reagents were obtained from Sigma or previously described sources (2, 4).

The irreversible Btk inhibitors BTKI-43607 and BTKI-43761 were provided by Pharmacyclics (Sunnyvale, CA), dissolved in DMSO at 10 mM, and stored at −20°C. BTKI-43607 and BTKI-43761 are analogs of ibrutinib and form a covalent bond with a cysteine residue on Btk. The molecular specificity of the interaction of BTKI-43607 and BTKI-43761 with Btk was evaluated using KinaseProfiler (Millipore). The IC₅₀ and selectivity of BTKI-43761 are reported in Table 1.

Table 1.

IC₅₀ values of the ibrutinib analog BTKI-43761

	BTKI-43761
Kinase	IC50, nM	Selectivity for Btk
Btk	0.39	1.0
ErbB4/HER4	0.64	1.6
Blk	0.94	2.4
Bmx/Etk	1.10	2.8
Fgr	2.86	7.3
Txk	2.87	7.4
Lck	3.49	9.0
Yes/YES1	3.94	10
Tec	5.49	14
Csk	6.17	16
EGFR	7.80	20
Brk	10.1	26
Itk	11.7	30
Hck	17.0	44
ErbB2/HER2	21.6	55
JAK3	21.9	56

Btk, Bruton's tyrosine kinase.

In vitro platelet studies.

Washed human platelets were prepared from venous blood drawn by venipuncture from healthy volunteers into sodium citrate, in accordance with a protocol approved by the Oregon Health & Science University Institutional Review Board; written informed consent was obtained from the human volunteers. The blood was centrifuged at 200 g for 20 min to obtain platelet-rich plasma (PRP). The platelets were isolated from PRP via centrifugation at 1,000 g for 10 min in the presence of prostacyclin (0.1 μg/ml). The platelets were then resuspended in modified HEPES-Tyrode buffer and washed once via centrifugation at 1,000 g for 10 min. Washed platelets were resuspended in modified HEPES-Tyrode buffer to the desired concentration. Static adhesion assay, Western blot, and flow cytometry experiments were performed as previously described (2, 4).

Platelet aggregation.

Platelet aggregation studies were performed using 300 μl of platelets (2 × 10⁸/ml) treated with inhibitors for 10 min. Platelet aggregation was triggered by CRP (3 μg/ml) or thrombin (0.1 U/ml) and monitored under continuous stirring at 1,200 rpm at 37°C by measuring changes in light transmission with a PAP-4 aggregometer, as previously described (4).

Platelet aggregate formation under flow.

Sodium citrate-anticoagulated blood was treated with inhibitors as indicated and perfused at 2,200 s⁻¹ at 37°C through glass capillary tubes coated with collagen (100 μg/ml) and surface-blocked with denatured BSA to form platelet aggregates, as previously described (3). Aggregate formation was imaged using Köhler-illuminated Nomarski differential interference contrast optics with a Zeiss ×400/0.75 NE EC Plan-Neofluar lens on a Zeiss Axiocam MRm camera and Slidebook 5.0 software (Intelligent Imaging Innovations). For computation of aggregate formation, platelet aggregates were manually outlined and quantified as previously described (3).

Nonhuman primate studies.

Nonhuman primate, male baboons (Papio anubis) were cared for and housed at the Oregon National Primate Research Center (ONPRC) at the Oregon Health & Science University. All the experiments described here were reviewed and approved (approval nos. IS00002496 and IS00002092) by the Oregon Health & Science University West Campus Animal Care and Use Committee according to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (ISBN 0-309-05377-3, 1996). The ONPRC is a category I facility. The Laboratory Animal Care and Use Program at the ONPRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care and has an approved assurance (no. A3304-01) for the care and use of animals on file with the Office for Protection from Research Risks at the National Institutes of Health.

To determine the effect of Btk inhibitors on platelet function in vivo, BTKI-43607 and BTKI-43761 were orally administered to individual nonhuman primates (n = 2) for 3 days at 10 mg·kg⁻¹·day⁻¹ and allowed to rest for 5 days. This dose was selected to test the maximal response and potential bleeding risk of these new ibrutinib analogs within the dose range of 1.25–12.5 mg·kg⁻¹·day⁻¹ used in clinical studies of ibrutinib (19). At regular intervals, blood was drawn into sodium citrate, and PRP was obtained via centrifugation of whole blood at 200 g for 8 min. Supernatant was removed, and platelet-poor plasma was obtained by further centrifugation of the remaining blood at 5,000 g for 5 min. Platelets were counted using a multispecies hematology system (Hemavet HV950). Platelet count in PRP was further adjusted to 2 × 10⁸/ml with platelet-poor plasma. Platelet aggregations were performed using the agonist CRP (1 and 0.5 μg/ml) in an aggregometer (Chrono-Log).

Next, a longer-time-course experiment was performed in which BTKI-43607 and BTKI-43761 were orally administered daily to individual nonhuman primates (n = 2) for 10 days at 10 mg·kg⁻¹·day⁻¹. Blood was withdrawn at regular intervals and processed as described above for platelet aggregation studies. Tests of prothrombin time (PT) and activated partial thromboplastin time (APTT) were also performed on blood samples. To test the effect of the Btk inhibitors on bleeding, a standard template skin bleeding time (BT) assessment was performed using a US Food and Drug Administration-approved incision device (Surgicutt, International Technidyne, Edison, NJ) at baseline and within 3 h of each treatment. Additionally, tourniquet test (capillary resistance test) studies, designed to detect abnormalities in capillary walls or thrombocytopenia, were performed. The bleeding assay, an indicator of overall hemostatic response, was performed in light of the fact that bleeding side effects have been seen in patients taking ibrutinib.

Statistical analysis.

For flow chamber experiments, data were fitted to the quasi-binomial distribution with the identity link function. For static adhesion and flow cytometry experiments, two-way ANOVA (with treatment and donor as factors) was followed by post hoc analysis with Tukey's test. For all tests, P < 0.05 was considered statistically significant. Statistical analyses were performed using R (R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Effect of Btk inhibitors on tyrosine phosphorylation in human platelets.

Btk has been shown to play a role in the regulation of tyrosine kinase activation downstream of the ITAM-coupled receptor GPVI in human platelets (26). Btk was rapidly phosphorylated following stimulation of purified platelets with the GPVI agonist CRP in a concentration- and time-dependent manner (Fig. 1, A and B). We next designed experiments to validate the effects of Btk inhibition on signaling downstream of GPVI. Pretreatment of platelets with the selective Src-family kinase inhibitor protein phosphatase 2 (PP2) abrogated Btk phosphorylation in response to 1 μg/ml CRP (Fig. 1C). Pretreatment of platelets with BTKI-43761 or BTKI-43607 also inhibited Btk phosphorylation in response to 1 μg/ml CRP (Fig. 1C). Interestingly, a reduction in Lyn phosphorylation was observed in the presence of BTKI-43761. The level of Syk phosphorylation was unaffected by pretreatment of platelets with 1 μM BTKI-43607 or BTKI-43761, while Syk phosphorylation was abrogated in the presence of PP2 (data not shown).

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.

Effect of Btk inhibitors on human platelet P-selectin membrane exposure.

Platelet activation results in surface exposure of the transmembrane protein P-selectin, which is stored in the membrane of platelet α-granules and is translocated to the platelet surface upon α-granule release (12). We next examined the effect of Btk inhibition on P-selectin exposure in response to platelet activation with the GPVI agonist CRP. As shown in Fig. 2, pretreatment of purified platelets with 1, 3, and 10 μM BTKI-43607 or BTKI-43761 eliminated P-selectin exposure in response to stimulation with 10 μg/ml CRP, as determined by flow cytometry measurement of platelet staining with an anti-P-selectin (CD62P) antibody. A similar degree of inhibition was observed in the presence of the Src kinase inhibitor PP2. In contrast, P-selectin exposure in response to platelet activation with the G protein-coupled agonist thrombin was unaffected by treatment of platelets with 1 or 3 μM BTKI-43607 or BTKI-43761, while a partial inhibition was observed by treatment with 10 μM BTKI-43761.

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.

Effect of Btk inhibitors on human platelet spreading on fibrinogen and collagen surfaces.

We next examined the role of Btk on the ability of platelets to spread on surfaces of CRP, a GPVI agonist, fibrinogen, which supports platelet spreading via α_IIbβ₃-integrin, or collagen, which supports platelet activation downstream of GPVI and adhesion and spreading via α₂β₁ and α_IIbβ₃ integrin (24). As seen in Fig. 3, treatment of platelets with 10 μM BTKI-43761 inhibited platelet spreading on CRP and on fibrinogen and collagen in the presence of the ADP scavenger apyrase, while BTKI-43607 failed to significantly inhibit platelet spreading on any of the three surfaces. Platelet spreading on CRP and collagen was inhibited by the Src kinase inhibitor PP2.

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.

Effect of Btk inhibitors on human platelet aggregate formation under flow.

We next examined the role of Btk in platelet adhesion and aggregate formation on collagen under shear flow conditions. Sodium citrate-anticoagulated whole human blood was pretreated with vehicle (DMSO), BTKI-43607, or BTKI-43761 before it was perfused over a surface of fibrillar collagen at a shear rate of 2,200 s⁻¹ (representative shear of stenosed arteries) for 4 min, fixed, and visualized. The extent of surface area coverage was analyzed for at least three fields of view for three independent experiments. As seen in Fig. 4, pretreatment of whole blood with 1 μM BTKI-43607 or BTKI-43761 significantly reduced the degree of platelet aggregate formation on collagen under shear, as measured by the extent of surface coverage of platelet aggregates, with a reduction of 43% and 17%, respectively, compared with control.

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.

Effect of Btk inhibitors on nonhuman primate platelet aggregation.

We next investigated the in vivo effect of Btk inhibition on nonhuman primate platelet aggregation. BTKI-43607 or BTKI-43761 was orally administered to nonhuman primates for 3 days at 10 mg·kg⁻¹·day⁻¹, and the animals were allowed to rest for 5 days. Our data demonstrate robust aggregation of nonhuman primate platelets in response to 1 μg/ml CRP prior to administration of BTKI-43607 or BTKI-43761 (Fig. 5, baseline), along with a change in platelet shape in response to 0.5 μg/ml CRP (data not shown). A dramatic inhibition of CRP-induced aggregation was observed as early as 1 h following administration of BTKI-43607 or BTKI-43761 (Fig. 5, day 0). Platelet aggregation was abrogated within 4 h after administration of BTKI-43607 or BTKI-43761 (day 0), and platelets failed to aggregate in response to CRP during the 3 days of BTKI-43607 or BTKI-43761 (days 0–2) treatment. While minimal aggregation was observed following 2 days of rest (day 4), platelet aggregation was restored to near-baseline levels by day 7.

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.

We next performed a time-course experiment in which BTKI-43607 was administered daily to the nonhuman primates for 10 days (10 mg·kg⁻¹·day⁻¹ po), followed by an 11-day nondosing period, followed by administration of BTKI-43761 for 10 days (10 mg·kg⁻¹·day⁻¹). Our data show that, prior to administration of BTKI-43607 or BTKI-43761, a robust degree of platelet aggregation in PRP was observed in response to 1 μg/ml CRP (Fig. 6, Table 2; day −3), and platelet shape change was observed in response to 0.5 μg/ml CRP (data not shown). Platelet aggregation and shape change were eliminated in response to 1 and 0.5 μg/ml CRP, respectively, during the entire time course of treatment with BTKI-43607 or BTKI-43761 (Fig. 6 and Table 2, days 0, 2, 4, and 9; data not shown).

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.

Table 2.

Average vertical drop and percent aggregation of platelet-rich plasma stimulated with CRP from nonhuman primates dosed with BTKI-43607 or BTKI-43761 in the long-term study

Day	Vertical Drop, cm	Percent Aggregation
No drug
−3	3.85 ± 0.05	100.0 ± 0.0
BTKI-43607
0	−0.95 ± 0.15	−24.6 ± 3.6
2	−0.15 ± 0.15	−3.9 ± 3.9
4	−1.00 ± 0.10	−26.0 ± 2.9
9	−1.00 ± 0.10	−25.9 ± 2.3
No drug
−3	3.85 ± 0.05	100.0 ± 0.0
BTKI-43761
0	−1.00 ± 0.20	−25.9 ± 4.9
2	−1.05 ± 0.15	−27.3 ± 4.3
4	−0.85 ± 0.05	−22.1 ± 1.0
9	−0.45 ± 0.15	−11.6 ± 3.7

Values are means ± SE (n = 2). Collagen-related peptide (CRP) was administered at 1 μg/ml.

Effect of Btk inhibitors on BT and capillary fragility in vivo.

To test the potential effect of Btk inhibitors on bleeding, a standard template skin BT assessment was performed using a US Food and Drug Administration-approved incision device (Surgicutt) at baseline and within 3 h of BTKI-43761 or BTKI-43607 administration. Our data show that neither BTKI-43761 nor BTKI-43607 significantly increased template BT during the 10-day dosing time course (Table 3). Moreover, tourniquet test (capillary resistance test) studies, designed to detect abnormalities in capillary walls or thrombocytopenia, were negative. In comparison, studies have shown that treatment of the nonhuman primates with 1,006 μg/kg low-molecular-weight heparin increased template BT by 1.46-fold over baseline (5).

Table 3.

PT, APTT, bleeding time, and CRT results from nonhuman primates dosed with BTKI-43607 or BTKI-43761 in the long-term study

	Dosing Day	PT, s	APTT, s	Bleeding Time, min	CRT
	Nonhuman primate A
No drug	−3	13.3	39.4	3	Negative
BTKI-43607 (10 mg/kg)	0	12.6	41.3	3	Negative
	2	12.5	38.8	3	Negative
	4	13.0	40.7	4	Negative
	9	13.8	38.8	3	Negative
BTKI-43761 (10 mg/kg)	0	12.8	37.5	3	Negative
	2	12.8	39.8	3	Negative
	4	12.6	38.7	4	Negative
	9	12.8	40.5	3	Negative
	Nonhuman primate B
No drug	−3	13.3	38.1	3	Negative
BTKI-43607 (10 mg/kg)	0	12.9	39.6	3	Negative
	2	12.5	39.6	3.5	Negative
	4	12.9	38.5	4	Negative
	9	12.4	38.3	3	Negative
BTKI-43761 (10 mg/kg)	0	13.2	37.9	3	Negative
	2	12.9	40.0	3.5	Negative
	4	13.1	38.3	3.5	Negative
	9	13.0	38.6	3.5	Negative

PT, prothrombin time; APTT, activated partial prothromboplastin time; CRT, capillary resistance test.

Effect of Btk inhibitors on coagulation.

The effect of Btk inhibitors on coagulation in vivo was evaluated by measurement of PT and APTT. Our data show that neither BTKI-43761 nor BTKI-43607 significantly increased plasma PT or APTT during the 10-day dosing time course (Table 3). In comparison, Atkinson et al. (5) showed that treatment of the nonhuman primates with 1,300 μg/kg low-molecular-weight heparin increased APTT by 1.9-fold over baseline.

DISCUSSION

This study examined the role of Btk in human platelet function in vitro and in vivo and nonhuman primate platelet function in vivo. The data demonstrate that inhibition of Btk reduced GPVI-mediated platelet activation, Btk phosphorylation, spreading on fibrinogen, and aggregation under shear flow. First, we confirmed that inhibition of Btk reduced Btk phosphorylation in CRP-stimulated human platelets but did not affect Syk phosphorylation compared with the positive control PP2, confirming that Btk acts downstream of Syk in the GPVI-ITAM signaling cascade. However, BTKI-43761 was also shown to partially inhibit Lyn phosphorylation, suggesting that this ibrutinib analog may have off-target effects on Src, Syk, or Tec family kinases. For instance, kinases with a cysteine in the active site, such as Tec, represent possible targets for covalent binding with ibrutinib analogs. Moreover, our kinase screen demonstrated that BTKI-43761 may partially inhibit Fgr, although recent work has shown that Fgr plays only a minor role in platelet signal transduction (23).

Studies of platelet activation by flow cytometry showed that inhibition of Btk eliminated P-selectin exposure in CRP-stimulated human platelets, supporting the notion that Btk plays a major role in platelet α-granule secretion via the GPVI pathway. These results are supported by early findings that the platelets of Btk-deficient XLA patients inhibited aggregation by CRP or, to a lesser extent, collagen, but not thrombin (21).

To explore platelet activation on immobilized surfaces or fibrinogen or collagen, static adhesion studies were performed. Platelet lamellipodia formation was significantly reduced on CRP, fibrinogen, and collagen surfaces in the presence of 10 μM BTKI-43761 and the ADP scavenger apyrase, suggesting that inhibition of Btk has an inhibitory effect on GPVI and α_IIbβ₃ integrin outside-in signaling, perhaps by inhibiting Btk activation of PLCγ2 downstream of ITAM signaling initiated by GPVI or activated α_IIbβ₃ integrin, respectively (16). Conversely, when ADP signaling was not blocked with apyrase, inhibition of platelet spreading on fibrinogen and collagen by 10 μM BKTI-43761 was reversed, suggesting that platelet activation through GPVI and integrin signaling coupled with ADP signaling via P2Y₁₂/P2Y₁ receptors was able to overcome Btk inhibition under static conditions. This highlights some redundancy within the ITAM- and G protein-coupled receptor-signaling pathways, which has been documented in knockout mouse studies showing that, following platelet activation by collagen, Tec kinase could partially compensate for Btk deletion under static conditions (5). These findings are in accord with the recent work by Bye et al. (9) demonstrating that ibrutinib inhibited platelet adhesion and spreading on CRP but not collagen under static conditions.

Platelet recruitment and aggregation under shear are mediated by the transient binding of vWF to platelet GPIb-GPIX-GPV, enabling the more durable adhesion of α₂β₁ integrin to collagen and subsequent activation of platelets via GPVI-mediated signaling, followed by platelet α_IIbβ₃ integrin-mediated aggregation. Our data show that inhibition of Btk partially reduced platelet aggregate formation on collagen under arterial rates of shear. Studies in Btk knockout mice have similarly found a vWF- and GPIb-dependent role for Btk signaling in collagen-induced platelet activation under arterial shear (17), supporting the findings of the current study, which utilized human platelets. These results are supported by recent investigations in which collagen-mediated adhesion and aggregation were significantly reduced under arterial shear in PRP of patients treated with the Btk inhibitor ibrutinib compared with controls (14, 15) and by studies showing an additive effect of the Btk inhibitor ibrutinib and the P2Y₁₂ inhibitor cangrelor on thrombus formation under shear flow (9).

Finally, a short-term study in nonhuman primates of the ibrutinib analogs BTKI-43607 and BTKI-43761 revealed a robust inhibition of GVPI agonist-induced aggregation for either inhibitor (10 mg/kg) by 4 h after administration. The impaired aggregation began to recover 2 days after dosing ceased, with full recovery of aggregation after 5 dose-free days. These data demonstrate that both ibrutinib analogs exhibit strong inhibition of aggregation. A longer trial with nonhuman primates showed that both BTKI-43607 and BTKI-43761 consistently inhibited GVPI agonist-induced platelet aggregation during a 10-day dosing period. Tests for bleeding, capillary abnormalities, and thrombocytopenia were negative throughout the dosing period for these ibrutinib analogs, revealing no measurable impairment of hemostasis during the study. These findings are in line with findings from the ibrutinib clinical trials, which reported predominantly low-grade bleeding events in humans (10, 25).

In conclusion, this study demonstrated that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro and in vivo, which may affect hemostasis in patients. However, bleeding side effects were not observed in a nonhuman primate model of template bleeding in this study.

GRANTS

This work was supported by National Institutes of Health Grants R01 HL-101972 (to O. J. T. McCarty) and T32 AI-007472 (to L. D. Healy), American Heart Association Grants 13POST13730003 (to J. E. Aslan) and 13EIA12630000 (to O. J. T. McCarty), and the Oregon Clinical and Translational Research Institute (National Institutes of Health Grant UL1 RR-024140 to L. D. Healy). M. L. D. Thierheimer is an Oregon State University Johnson Scholar. R. A. Rigg is a Whitaker International Fellow.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.A.R., J.E.A., M.T.H., A.G., and O.J.T.M developed the concept and designed the research; R.A.R., J.E.A., L.D.H., M.W., M.L.D.T., C.P.L., and J.P. performed the experiments; R.A.R., J.E.A., L.D.H., C.P.L., and J.P. analyzed the data; R.A.R., J.E.A., M.T.H., A.G., and O.J.T.M interpreted the results of the experiments; R.A.R. and J.E.A. prepared the figures; R.A.R., J.E.A., and O.J.T.M drafted the manuscript; R.A.R., J.E.A., M.T.H., A.G., and O.J.T.M edited and revised the manuscript; R.A.R., J.E.A., L.D.H., M.W., M.L.D.T., C.P.L., J.P., M.T.H., A.G., and O.J.T.M approved the final version of the manuscript.