Ibrutinib, but not zanubrutinib, induces platelet receptor shedding of GPIb-IX-V complex and integrin α_IIbβ₃ in mice and humans

Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (Brk), a member of the Tec family that is essential in B-cell antigen receptor signaling.^1-3  Ibrutinib is approved for treatment of patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and Waldenström macroglobulinemia.^3-8  Ibrutinib is also improved for marginal zone lymphoma and graft-versus-host disease in the United States.^3-8  Although ibrutinib is generally well tolerated, clinical studies have reported grade ≥3 bleeding events, including hematomas, hematuria, and gastrointestinal bleeding in 5% to 6% of patients.⁹  This bleeding risk is increased by concurrent therapy with antiplatelet agents or anticoagulants, including warfarin and direct oral anticoagulants.⁹  This is a particular problem in the elderly, who often have concurrent antithrombotic therapy and who are underrepresented or excluded from clinical trials.⁹ 

Btk plays an essential role in the GPIb and GPVI signaling pathways, which mediate adhesion to von Willebrand factor (VWF) and collagen, particularly in high shear rates in vivo.^10-14  Patients with X-linked agammaglobulinemia do not have bleeding problems, despite the absence of functional Btk.¹⁵  This is consistent with the observation that the second-generation Btk inhibitors acalabrutinib and zanubrutinib have been reported to have lower bleeding rates compared with ibrutinib in clinical trials. This finding suggests that off-target effects of ibrutinib may contribute to bleeding.^16-18  Zanubrutinib has a more specific targeted binding profile than ibrutinib with less off-target effects on enzymes, including tyrosine kinase expressed in hematopoietic carcinoma, human epidermal growth factor receptor 2, Janus kinase 3 (JAK3), epidermal growth factor receptor, and interleukin 2-inducible T-cell kinase.¹⁹  Previous studies have demonstrated that ibrutinib affects collagen and 4 VWF-dependent platelet functions but not G-protein coupling–dependent functions.¹²  In addition, ibrutinib inhibits platelet integrin α_lIbβ₃ outside-in signaling and thrombus stability, but not adhesion to collagen.²⁰  These findings suggest that ibrutinib therapy is associated with complex effects on multiple platelet signaling pathways that contribute to bleeding events.

Platelet receptor internalization and/or shedding is thought to be important in clearing aged platelets from the circulation and regulates surface expression of a range of glycoproteins, including GPIb-IX-V complex and GPVI.²¹  Receptor shedding is induced by agonists and ADAM family members. GPIbα is exclusively shed by agonist-induced activation of ADAM17 protease, resulting in generation of soluble GPIbα fragments. In contrast, collagen GPVI receptor is normally shed by agonist-induced activation of ADAM10 protease.^21,22 

In the present study, we investigated the effects of ibrutinib compared with a second-generation Btk inhibitor, zanubrutinib, on platelet function, glycoprotein expression, and thrombus formation in blood flow, by using platelets from C57BL/6 mice, healthy donors, and Btk inhibitor–treated patients with CLL. Ibrutinib-treated but not zanubrutinib-treated blood samples from healthy donors or Btk inhibitor-treated patients with B-CLL, demonstrated reduced thrombus formation on immobilized collagen, VWF, or fibrinogen in arterial flow conditions. Our experiments further showed that ibrutinib but not zanubrutinib treatment resulted in loss of GPIb-IX-V complex from the platelet surface in an ADAM17- but not ADAM10-dependent manner. This reduction was observed for GPIb-IX-V complex expression but not GPVI expression on the platelets in healthy donors treated in vitro by ibrutinib and a proportion of chronically treated B-CLL patients. These data demonstrate that ibrutinib induces shedding of GPIbα surface receptor by an ADAM17-dependent mechanism and that the process occurs in vivo.

Materials are listed in the supplemental Information.

BGB-3111 (zanubrutinib; 97.60% purity) and BGB-1672 (ibrutinib; 98.75% purity) were supplied by Beigene. Ibrutinib was also extracted from capsules (Johnson and Johnson, North Ryde, NSW, Australia). All data analyses in this paper are based on the purified forms of ibrutinib and zanubrutinib.

All animal experiments and care were approved by the Royal Melbourne Institute of Technology (RMIT) University Animal Ethics Committee (AEC1628). C57BL/6 mice were used at 6 to 10 weeks of age.

All human Btk-treated B-CLL blood samples and experiments were approved by the Peter MacCallum Cancer Centre Human Research Ethics Committee (HREC), Project 18/206. Patient samples were from clinical trials BGB-3111-AU-003 and NCT02343120. All 5 ibrutinib-treated patients received commercial drug.

Platelet rich plasma (PRP) and washed human or murine platelets were isolated, and platelet aggregation experiments were performed as previously described.^23-25 

Full blood examination parameters were determined as previously described.^23-25 

Release of α and dense granules was performed as previously described.²³ 

Clot retraction was performed as previously described.^24,26 

Tail bleeding time was determined as previously described.²⁴ 

Washed platelets (50 µL of 100 × 10⁹/L) were preincubated with the PE-conjugated antibodies human CD42a (GPIX), anti-human CD41a (integrin α_IIb), anti-human CD61 (integrin β₃), anti-human CD42b (GPIbα), anti-human GPVI, or anti-human CD42c (GPIbβ); APC-conjugated anti-human CD42d (GPV) antibodies; or FITC-conjugated PAC-1 antibodies. The subsequent steps and analysis have been described.^23-25 

A 6-channel µ-Slide VI, with dimensions (0.1 × 1.0 × 17 mm, H × W × L; Microslides; Ibidi, Martinsried, Germany), was precoated with 500 µg/mL type I fibrillar collagen (Nycomed, Linz, Austria), 50 µg/mL VWF, or 100 µg/mL fibrinogen for 2 hours at 37°C, followed by washing with Tyrode’s buffer. Whole blood from healthy donor and Btk-treated CLL patients was normalized to 200 × 10⁹/L platelets and incubated with 0.05% (w/v) rhodamine 6G dye for 30 minutes at 37°C. Labeled platelets were then perfused through the collagen-coated microslide at an arterial shear flow rate of 1800 seconds⁻¹ for 6 minutes. Nonadherent cells were washed away with Tyrode’s buffer. Thrombi formation was recorded in real time over the 6-minute time frame using z-stack analysis and deconvolution with 3-dimensional (3D) reconstructions, using Zeiss Axiovert M1 microscope with AxioCam MRm camera (Zeiss, Thornwood, NY). Thrombus area (in square micrometers), thrombus height (micrometers), and thrombus volume (cubic micrometers) were determined at 2, 4, and 6 time points.

C57BL/6 mice were treated with ibrutinib (10 mg/kg), zanubrutinib (10 mg/kg), or dimethyl sulfoxide (DMSO) control for 2 hours. Citrated whole blood was obtained from C57BL/6 mice, and the platelets were fluorescently labeled with 0.05% (w/v) rhodamine 6G dye. After incubation at 37°C for 30 minutes, labeled platelets in whole blood were then perfused over a matrix of type I collagen–coated µ-Slide VI^0.1 for 6 minutes at a shear stress rate of 1800 seconds⁻¹. Thrombus formation was monitored as described above.

C57BL/6 mice (4-6 weeks old) were orally administered with the Btk inhibitors 10 mg/kg ibrutinib and 10 mg/kg zanubrutinib or DMSO-treated control by oral gavage. After 2 hours (peak drug concentration), Btk inhibitor–treated mice were anesthetized with a ketamine/xylazine (100:10 mg/kg) mixture intraperitoneally. Ferric chloride (FeCl₃) injury and intravital microscopy were performed as previously described.²³ 

Washed platelets (200 µL of 100 × 10⁹/L) were placed into a 96-well white plate (Perkin Elmer, Melbourne, VIC, Australia) and pretreated with 20 µM fluorogenic TACE substrate (7-methoxycoumarin-4-yl) acetyl-P LAQAV-N-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl-RSSSR-NH2) at 37°C for 20 minutes. Platelets were then incubated with vehicle control, type I collagen (20 µg/mL), ibrutinib (0.5 µM), or zanubrutinib (0.5 µM) for 40 minutes at 37°C. The cleavage of the TACE fluorogenic substrate was monitored with a Clariostar microplate reader (BMG Labtech, Mornington, VIC, Australia) set at 320/405 nm.

Soluble murine GPIbα (Cusabio, Houston, TX) and soluble murine α_IIb levels (Cusabio) were measured by solid phase sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Plasma was isolated from citrated whole blood collected from 8-week-old C57BL/6 mice treated with 10 mg/kg ibrutinib, 10 mg/kg zanubrutinib, or sham control for 2 hours. The samples were centrifuged at 1000g for 10 minutes to obtain platelet-poor plasma. All ELISA assays were read on a Victor X3 multilabel plate reader at 450 nm wavelength (Perkin Elmer, Waltham, MA).

Statistical analysis was performing using Prism software, version 7 (GraphPad, San Diego, CA). Data sets were expressed as the mean ± standard error of the mean (SEM). Statistical significance for 2 data sets was determined with the unpaired Student t test. A value of P ≤ .05 was considered significant.

Ibrutinib and zanubrutinib are specific Btk inhibitors that target the same cysteine residue on Btk to regulate ITAM-dependent pathways. To examine the effect of ibrutinib and zanubrutinib on thrombus formation under high shear flow conditions in vitro, we incubated human blood with ibrutinib or zanubrutinib (0.5 µM) and studied platelet adhesion to type I collagen, VWF, or fibrinogen over time. Vehicle- and zanubrutinib-treated platelets (0.5 µM) adhered and formed stable thrombi over a 6-minute period. In contrast, ibrutinib-treated platelets (0.5 µM) dramatically reduced the number of adherent platelets and virtually abrogated thrombus formation, as shown by a significant reduction in thrombus volume on type I collagen matrix or VWF or fibrinogen over the 6-minute period (Figure 1). This indicated an antiplatelet mechanism induced by ibrutinib, but not by zanubrutinib, that directly interfered with platelet adhesion on collagen, VWF, and fibrinogen under conditions of arterial shear.

When platelets are stimulated, the processes of platelet activation are enhanced by α-granule release (P-selectin expression) and dense granule production of ADP and serotonin that make platelet aggregation more stable. To investigate the effect of Btk inhibitors (ibrutinib and zanubrutinib) on α- or dense-granule release, platelets pretreated with vehicle DMSO-treated or Btk inhibitor were stimulated with agonists, including thrombin (0.25 U/mL), PAR-1 (1.25 µM), PAR-4 (25 µM), CRP-XL (0.25 µg/mL), and rhodocytin (0.7 µg/mL). The concentration dependence of ibrutinib and zanubrutinib (0.5-2.0 µM) on agonist-induced α- and dense-granule exocytosis was examined. P-selectin expression was measured by flow cytometry with an FITC-labeled anti-CD62P antibody, whereas dense granule exocytosis was determined by quinacrine release. Ibrutinib or zanubrutinib significantly inhibited rhodocytin (CLEC-2 ligand)- and CRP-XL (GPVI selective agonist)–induced P-selectin expression and dense granule release at different doses of drugs (0.5-2.0 µM) (Figure 2A-J). In contrast, there was no effect of ibrutinib or zanubrutinib observed with thrombin, PAR-1–, or PAR-4–induced P-selectin expression and dense granule exocytosis. Ibrutinib but not zanubrutinib also inhibited PAC-1 binding (Figure 2K). These data indicate that both drugs inhibit GPVI and CLEC-1 hemi (ITAM)-mediated signaling pathways that modulate α- and dense-granule exocytosis.

We evaluated the effect of pretreatment of platelet-rich plasma (PRP; platelets normalized to 100 × 10⁹/L) with a dose-dependent range of concentrations of ibrutinib or zanubrutinib (0.25-1.0 µM; levels similar to those seen in the clinic) compared with DMSO-treated normal healthy human platelets on agonist-induced platelet aggregation responses as measured with a 4-channel platelet aggregometer. Consistent with the known role of Btk in GPVI signaling, ibrutinib and zanubrutinib inhibited CRP-XL, 1.0 µg/mL induced platelet aggregation at all doses compared with vehicle control. Ibrutinib and zanubrutinib also inhibited collagen-induced platelet aggregation when tested at 1.0 µg/mL, but to a lesser extent with zanubrutinib. Ibrutinib but not zanubrutinib also inhibited ADP- and TRAP-induced platelet aggregation at all drug doses tested (Figure 3A-D). This finding is in agreement with those in a previous study.²⁷ 

Integrin α_IIbβ₃ is crucial for normal platelet aggregation, platelet spreading, and clot retraction functionality. Therefore, we next examined whether there is any significant difference in the kinetics of the clot retraction process after Btk inhibitor treatment of PRP before addition of thrombin to induce clotting. Ibrutinib-treated PRP significantly delayed the kinetics of clot retraction over the 2-hour time frame, compared with zanubrutinib-treated PRP and vehicle control (Figure 3E-F).

Several platelet receptors that are involved in the adhesion and aggregation process, including GPIb-IX-V complex, integrin α_IIbβ₃, and GPVI, are proteolytically downregulated in response to various agonists.²⁴  Ibrutinib has been demonstrated to interfere with platelet adhesion on collagen but whether platelet glycoproteins are affected is not known. To investigate whether ibrutinib modulates the surface glycoprotein expression on platelets, we incubated whole blood with different concentrations of ibrutinib or zanubrutinib or vehicle control and measured the surface expression of the major glycoproteins on the platelet surface by flow cytometry. Ibrutinib but not zanubrutinib treatment of platelets caused a time- and dose-dependent downregulation of GPIbα, GPIX, and integrin α_IIbβ₃ (both α_IIb and β₃ subunits were reduced), but not GPVI, GPV, or GPIbβ (Figure 4A-K). This ibrutinib effect was observed with either a purified form of the drug or ibrutinib extracted from capsules that were given to patients with B-CLL (data not shown).

To characterize the mechanism underlying the ibrutinib-mediated loss of specific platelet glycoproteins and to identify the responsible metalloproteinase involved, we incubated whole human blood pretreated with GM6001, TAPI-2, or vehicle control before ibrutinib treatment. Pretreatment with the metalloproteinase inhibitor GM6001 or the ADAM17 inhibitor TAPI-2 before ibrutinib exposure blocked the loss of GPIbα and GPIX from the platelet surface (Figure 4L-N). In contrast, integrin α_IIbβ₃ was still lost from the platelet surface, even with pretreatment with GM6001 or TAPI-2. As ADAM17 is the exclusive metalloproteinase that mediates GPIbα and GPIX shedding from the platelet surface, these results suggest that ibrutinib mediates shedding of GPIbα, and GPIX occurs in an ADAM17-dependent manner.

To further characterize the mechanism underlying the ibrutinib-mediated loss of integrin α_IIbβ₃ and GPIbα and to identify the potential responsible protease involved, we incubated whole human blood pretreated with the ADAM10 inhibitor GI254023, the calpain inhibitor calpeptin, the dynamin inhibitor Dynasore hydrate, or the integrin α_IIbβ₃ antagonist eptifibatide. Pretreatment with the ADAM10 inhibitor (an ADAM family member in platelets), the calpain inhibitor (calpain is known to cleave β₃ cytoplasmic domain in platelets), the dynamin inhibitor (to inhibit integrin α_IIbβ₃ internalization), or integrin α_IIbβ₃ antagonist (to inhibit integrin α_IIbβ₃ activation) did not block the loss of GPIbα or integrin α_IIbβ₃ from the platelet surface (Figure 4O-P). These results confirm that ibrutinib-mediated loss of GPIbα is specific to ADAM17 activity, and integrin α_IIbβ₃ is lost via an unknown sheddase in platelets.

We next examined the effect of Btk inhibitors on murine thrombus formation of type I collagen during arterial flow, using whole blood derived from C57BL/6 mice. Citrated whole murine blood was treated with ibrutinib (0.5 µM) or zanubrutinib (0.5 µM), compared with vehicle control, for 30 minutes at 37°C. Whole blood was fluorescently labeled with rhodamine 6G before perfusion through a collagen-coated microcapillary tube flow chamber at an arterial shear rate of 1800 seconds⁻¹. After 6 minutes of sample perfusion, thrombus images for Btk- vs vehicle-treated whole blood were recorded in real time and analyzed by 3D reconstructions. Ibrutinib inhibited thrombus growth, as shown by reduced surface coverage and thrombus volume over time (Figure 5A-B). In contrast, zanubrutinib had thrombus growth characteristics similar to those of the vehicle control on type I collagen during arterial flow over time.

In ex vivo mouse models, various Btk inhibitors (ibrutinib 10 mg/kg or zanubrutinib 10 mg/kg) were administered orally. The dosing of ibrutinib and zanubrutinib of 10 mg/kg in in vivo mouse experiments is bioequivalent to that in Btk-treated CLL in patients.³  Whole blood was taken 2 hours later (peak drug concentration), rhodamine-labeled, then perfused onto immobilized type I fibrillar collagen at an arterial shear rate of 1800 seconds⁻¹. Thrombus images were recorded in real time over 6 minutes of blood perfusion. Thrombus volume was significantly inhibited by ibrutinib treatment (10 mg/kg) compared with equivalent thrombus growth for zanubrutinib (10 mg/kg) and vehicle-treated control over time (Figure 5C-D). In addition, increased tail bleeding times, associated with an enhanced volume of blood loss and unstable hemostasis, were found in ibrutinib-treated mice but not in wild-type or zanubrutinib-treated mice (Figure 5G-I).

Using in vivo mouse models, various Btk inhibitors (ibrutinib 10 mg/kg or zanubrutinib 10 mg/kg) were administered orally. After 2 hours of incubation, mice were fully anesthetized before cannulation of the jugular vein and exteriorization of the mesenteric arterioles (80-100 µm) was performed. FeCl₃-induced vascular injury was undertaken to induce the formation of in vivo blood clots, which was monitored in real time by intravital microscopy. Ibrutinib-treated C57BL/6 mice had smaller thrombus growth over time compared with zanubrutinib- and vehicle control-treated C57BL/6 mice (Figure 5E-F).

To test whether ibrutinib can induce shedding of GPIbα and integrin α_IIbβ₃ in vivo, we dosed C57BL/6 mice with vehicle, ibrutinib (10 mg/kg), or zanubrutinib (10 mg/kg) by oral gavage for 2 hours, and platelets derived from whole blood were then labeled with anti-mouse CD42b (GPIbα) or anti-mouse CD41 (integrin α_IIbβ₃) antibodies and analyzed by flow cytometry. Ibrutinib induced loss of GPIbα and integrin α_IIbβ₃ from murine platelet surfaces, whereas zanubrutinib was comparable to the vehicle control (Figure 6A-B). In addition, plasma levels of shed GPIbα and integrin α_IIbβ₃ increased 2- and 1.5-fold, respectively, compared with vehicle- or zanubrutinib-treated mice (Figure 6C-D). This result demonstrated that ibrutinib induces shedding of GPIbα and integrin α_IIbβ₃ in vivo.

As ibrutinib is associated with shedding of platelet glycoproteins in vivo, we next investigated whether patients with B-CLL treated with ibrutinib had reduced surface expression of GPIbα and integrin α_IIbβ₃ and ex vivo thrombus formation on type I collagen compared with zanubrutinib-treated patients or healthy controls. Table 1 summarizes information of the Btk inhibitor–treated patients. GPIb-IX-V complex expression on platelets derived from ibrutinib-treated patients with CLL had statistically significant lower levels compared with healthy controls or untreated or zanubrutinib-treated patients (Figure 7A-C). In addition, integrin α_IIbβ₃ expression on platelets derived from ibrutinib-treated patients with CLL had statistically significant lower levels compared with healthy controls, untreated patients, or zanubrutinib-treated patients. In contrast, platelets derived from ibrutinib-treated patients had similar levels of GPVI expression compared with those from healthy controls, untreated CLL patients, or zanubrutinib-treated patients. There was an exception of 1 zanubrutinib-treated patient with lower GPVI expression. All assessments were performed with normalized platelets from Btk inhibitor–treated patients with B-CLL vs healthy controls. Ibrutinib-treated B-CLL platelets formed smaller thrombi over time compared with zanubrutinib-treated, untreated B-CLL, and healthy normal control platelets (Figure 7D-E). Thrombus volume was reduced in ibrutinib-treated B-CLL platelets at different time points compared with zanubrutinib-treated, untreated, and healthy normal control B-CLL platelets, which showed similar thrombus volume over time. Taken together, these data indicate that ibrutinib- but not zanubrutinib-treated B-CLL patients had reduced thrombus growth on type I collagen, demonstrating an effect of ibrutinib on ex vivo platelet function.

To investigate why ibrutinib-induced shedding of GPIbα and GPIX occurs in an ADAM17-dependent manner, we used a TACE substrate to examine the effect of ibrutinib compared with zanubrutinib and vehicle control on ADAM17 TACE activity in vitro. Ibrutinib treatment of washed platelets resulted in increased TACE activity in vitro compared with the effect of zanubrutinib or vehicle control treatment (Figure 7F).

Before our study, it was thought that ibrutinib inhibits a combination of collagen GPVI signaling and outside-in integrin α_IIbβ₃ signaling to reduce the platelet function and thrombus formation that contribute to bleeding risk.^16,20  Here, we report that ibrutinib induces the proteolytic cleavage of human and murine platelet surface receptors, including GPIbα, GPIX, and integrin α_IIbβ₃ in vitro and in vivo. In the case of GPIbα, ibrutinib increases activation of the metalloproteinase ADAM17 and mediates proteolytic cleavage of GPIbα and GPIX, but not of GPV or GPIbβ. In contrast, integrin α_IIbβ₃ is shed by an unknown protease. These processes are relevant in vivo, because mice given ibrutinib by oral gavage had reduced ex vivo and in vivo thrombus formation, prolonged tail bleeding times, and increased levels of shed GPIbα and platelet-specific integrin α_IIb in their plasma.

In vitro thrombus formation experiments demonstrated that ibrutinib reduced platelet adhesion and thrombus formation on immobilized type I collagen, VWF, and fibrinogen. These adhesive interactions are involved in direct collagen and indirect collagen receptors, GPIb-IX-V complex, and integrin α_IIbβ₃. This was not the case for the more selective Btk inhibitor zanubrutinib, and it raised the possibility that ibrutinib modulates platelet adhesion and thrombus formation by a different mechanism, apart from modulating GPVI signaling or outside-in integrin α_IIbβ₃ signaling in platelets. We therefore investigated the surface expression of platelet glycoproteins in vitro and in vivo and found that ibrutinib treatment induced loss of GPIbα, GPIX, and integrin α_IIbβ₃, but not of GPVI, GPV, or GPIbβ. This raised the possibility that either proteolytic shedding or internalization occurs in vitro or in vivo. To address these possibilities, we examined a range of inhibitors, including the broad-spectrum metalloproteinase inhibitor GM6001, the ADAM17 inhibitor TAPI-2, the ADAM10 inhibitor GI254023, the calpain inhibitor calpeptin, the dynamin inhibitor Dynasore hydrate, and the integrin α_IIbβ₃ antagonist eptifibatide, to see whether they would block the loss of either GPIbα or integrin α_IIbβ₃ from the platelet surface. Our results showed that the ADAM17 inhibitor TAPI-2 blocked the ibrutinib-induced shedding of GPIbα and GPIX from the platelet surface. This was not the case with the ADAM10 inhibitor GI254023, the calpain inhibitor calpeptin, the dynamin inhibitor Dynasore hydrate, and the integrin α_IIbβ₃ antagonist eptifibatide. In contrast, the ADAM17 inhibitor TAPI-2, the ADAM10 inhibitor GI254023, the calpain inhibitor calpeptin, or the integrin α_IIbβ₃ antagonist eptifibatide did not block the ibrutinib-induced shedding of integrin α_IIbβ₃ from the platelet surface.

The mechanism by which ibrutinib exerts its action is mainly attributable to an off-target effect where metalloproteinase ADAM17 activity is increased by the protease’s being quickly switched on when platelets are activated to induce proteolytic shedding of GPIbα from the platelet surface. In addition, ibrutinib appears to affect an unknown sheddase to induce proteolytic shedding of integrin α_IIbβ₃ from the platelet surface. This hypothesis is based on the fact that zanubrutinib targets Btk as potently as ibrutinib but appears to be more selective in its Btk inhibition without the observed effects on platelet function and receptor shedding.

In platelets, the predominant sheddases are ADAM10 and ADAM17, with ADAM17 showing significant effects on a range of platelet receptors including GPIX and, exclusively, GPIbα and GPV.^21,22  ADAM10 activity plays an important role in downregulating GPVI levels on platelets.²¹  Under physiological conditions, GPIbα is constitutively shed as a glycocalicin fragment, with 2 µg/mL present in human plasma, and is thought to be important as a measure of platelet turnover.²¹  High-dose aspirin treatment has been shown to induce ADAM17-mediated GPIbα shedding in vitro and in vivo.²⁸  The significance of this process is not well understood, but very likely indicates downregulation of the inflammatory response and platelet responsiveness.

In this study, we demonstrated that ibrutinib, a Btk inhibitor, appears to have off-target effects that induce ADAM17-mediated shedding of GPIbα and GPIX in vitro and of GPIbα in vivo, as a soluble glycocalicin fragment detected in mouse plasma. This was not the case with the more selective Btk inhibitor zanubrutinib. The shedding process was not dependent on ADAM10 or calpain cleavage, nor was it regulated by outside-in integrin α_IIbβ₃ signaling. The mechanism underlying this ibrutinib effect is not completely clear, but it was shown that ibrutinib treatment increased ADAM17 TACE activity compared with zanubrutinib and vehicle control in vitro. Under physiological conditions, proteolytic cleavage of the prodomain activates the ADAM family members; however, direct activation of ADAM sheddases can occur with thiol-modifying agents or calmodulin inhibitors to induce GPIbα shedding in vitro.²⁹  The precise mechanism of how ibrutinib induces GPIbα and GPIX shedding requires further investigation, but the increased activation of ADAM17 may be related to the off-target effects of ibrutinib on 1 or more kinases, including protein kinase C, p38 kinase, or pololike kinase. Alternatively, ibrutinib is known to have potential off-target effects with 9 other cysteine-containing kinases in the human kinome that have cysteine at a similar position within the ATP binding pocket (Bmx, Tec, Txk, Itk, EGFR, Erb2, Erb4, Jak3, and Blk).

Apart from GPIbα, in this study ibrutinib also induced shedding of integrin α_IIbβ₃ from the surface of platelets in vitro and in vivo. However, in contrast to GPIbα and GPIX, it was independent of ADAM17-, ADAM10-, or calpain-induced shedding and was not regulated by outside-in integrin α_IIbβ₃ signaling. Analysis of the platelet sheddome highlighted that integrin α_IIb and β₃ subunits were detected in the supernatant of activated platelets, indicating that they can be shed from the surface of platelets.²²  The mechanism of how this shedding process occurs with integrin α_IIbβ₃ is unknown. Previous studies have highlighted a role for calpain cleavage of the intracellular cytoplasmic domain of integrin β₃, and high shear forces can induce shedding of integrin α_IIbβ₃.^30,31  Although ADAM17 has been shown to have mild effects on integrin α_IIbβ₃, it was excluded as a possible mechanism in the current study.²²  Whereas calpain cleavage may have also contributed to ectodomain shedding, calpeptin treatment before ibrutinib treatment did not block loss of integrin α_IIbβ₃ from the platelet surface.

The likely explanation for the loss of integrin α_IIbβ₃ from the surface of platelets after ibrutinib treatment may be attributable to induction of internalization or shedding of the receptor. Blocking internalization with a dynamin inhibitor, Dynasore hydrate before ibrutinib treatment did not reverse the ibrutinib-mediated reduction in integrin α_IIbβ₃ surface expression. However, in our mouse model, we were able to demonstrate that platelet-specific soluble integrin α_IIb was present in increased amounts in plasma after exposure to ibrutinib in vivo compared with zanubrutinib and vehicle control, supporting the concept that shedding is associated with loss of integrin α_IIbβ₃ from the platelet surface. These experiments highlighted that ibrutinib treatment induces integrin α_IIbβ₃ shedding from the platelet surface by an unknown sheddase. Further studies are needed to determine the exact sheddase involved in integrin α_IIbβ₃ ectodomain shedding in vivo.

Apart from in vitro and in vivo experiments, we also examined B-CLL patient samples from untreated or ibrutinib- vs zanubrutinib-treated and normal healthy controls and showed a significant proportion of ibrutinib-treated samples but not zanubrutinib or healthy controls had reduced surface expression of GPIbα, GPIX, and integrin α_IIbβ₃. In addition, we demonstrated that ibrutinib-treated patients with B-CLL had reduced ex vivo thrombus formation on type I collagen during arterial flow compared with zanubrutinib-treated patients, who were equivalent to untreated patients and normal healthy controls. This finding indicates that the shedding of major platelet receptors affects platelet function in ibrutinib-treated B-CLL patients and can explain why these patients are at risk of major bleeding. In this context, for patients with B-CLL, zanubrutinib may be safer in terms of bleeding risk for treating their hematological malignancy while maintaining a primary hemostatic balance with milder adverse events of petechiae/bruising reported in 38% of cases,³²  whereas acalabrutinib is associated with bleeding events in ∼50% of patients with hematological malignancies.³³  This is most likely attributable to on-target inhibition of collagen GPVI signaling pathways.

Other workers have studied the effect of ibrutinib on human platelets in modulating collagen- and VWF-dependent functions¹²  and integrin α_IIbβ₃ outside-in signaling.²⁰  We also observed that ibrutinib affected collagen- and VWF-dependent functions and outside-in integrin α_IIbβ₃ signaling readouts. However, our study showed that a quantitative reduction in GPIb-IX and integrin α_IIbβ₃ associated with shedding mediated by ibrutinib treatment contributed to the qualitative functional abnormalities observed in these previous studies.

In summary, in the current study, ibrutinib, but not zanubrutinib, caused shedding of human and mouse GPIbα, and GPIX in vitro and in vivo from the platelet surface. This process involves increased activation of the metalloproteinase ADAM17 and is exclusively associated with ADAM17. In addition, we have shown that ibrutinib but not zanubrutinib induces shedding of human and mouse integrin α_IIbβ₃ in vitro and in vivo by an unknown sheddase. These results are important, as they are a like explanation of why patients with ibrutinib-treated B-CLL have a higher risk of bleeding complications. At this stage, zanubrutinib is not yet approved for use in the United States, and acalabrutinib has been approved by the US Food and Drug Administration only for mantle cell lymphoma. Until these more selective Btk inhibitors are approved in more settings, ibrutinib will continue to be the dominant Btk inhibitor in clinical use.

This work was supported by a sponsored research agreement from Beigene, Pty Ltd (D.E.J.); funding from CLL Global Research Foundation (C.T.); and a postgraduate scholarship from Jazan University and the Ministry of Higher Education (Riyadh, Saudi Arabia) (G.D.).

Contribution: G.D., F.A.K., M.M.A.O., F.A., and A.M.G. performed the experiments; D.M.-y.S., S.M.H., C.P.S.T., and C.T. provided intellectual input and reviewed the paper; and D.E.J. directed the research and wrote the manuscript.

Conflict-of-interest disclosure: Beigene Pty, Ltd, provides funding to C.T. and D.E.J. The remaining authors declare no competing financial interests.

Correspondence: Denise E. Jackson, Thrombosis and Vascular Diseases Laboratory, School of Health and Biomedical Sciences, RMIT University, P.O. Box 71, Bundoora, VIC 3083, Australia; e-mail: denise.jackson@rmit.edu.au.