Since the first description of a patient with recurrent infections and deficiency of immunoglobulins termed “Agammaglobulinemia” by Ogden Bruton in 1952 (1), it took more than 40 years of research, until Bruton tyrosine kinase (Btk) was identified in 1993 as the responsible protein that is deficient in patients with X-linked agammaglobulinemia (2, 3). Btk belongs to the Tec (tyrosine kinase expressed in hepatocellular carcinoma) family of non-receptor cytoplasmic tyrosine kinase, and contains five different protein interaction domains: an amino terminal pleckstrin homology (PH) domain, a proline-rich Tec homology (TH) domain, the SRC kinase homology (SH) domains SH2 and SH3, and a kinase domain (4). Btk is the best studied member of this tyrosine kinase family and most homologous to Tec, the namesake of this kinase family. Btk plays a crucial role in B-cell receptor function and in immunoglobulin Fc- receptor signaling of monocytes/macrophages and neutrophils (4). Since the approval of ibrutinib, the covalent irreversible first in class Btk inhibitor (BTKi) in 2013 for treatment of certain B-cell malignancies, many more reversible and irreversible BTKi have evolved and the spectrum of diseases that are targeted extends from specific forms of B-cell malignancies to various autoimmune disorders (5). Up to now four BTKi (ibrutinib, acalabrutinib, zanubrutinib, and tirabrutinib) have been approved but at least further eight BTKi are in clinical studies (5).

Btk is expressed not only in B-cells but also in various hematopoietic cells including the megakaryocyte-platelet lineage (6). Btk in platelets is involved in signaling of specific glycoprotein receptors including glycoprotein (GP) VI activation by collagen, GPIb activation by von Willebrand factor (VWF), FcγRIIa activation by IgG immune complexes, and CLEC-2 activation by podoplanin (5). Thus, Btk might be a promising therapeutic target of platelet-related diseases, and BTKi have indeed been proposed as novel antiplatelet drugs as they inhibit selectively platelet GPVI/GPIb-stimulated atherothrombosis (7, 8), platelet FcγRIIA-dependent immune disorders (heparin-induced thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia) (9, 10), and podoplanin/CLEC-2 mediated venous thrombosis (11, 12). Somewhat surprisingly, XLA patients do not show a bleeding tendency (13). In contrast, mild bleeding events are frequent in patients with B-cell malignancies treated with irreversible covalent BTKi (ibrutinib, acalabrutinib, zanubrutinib, and tirabrutinib) (5). The reasons are not clear but are probably multifactorial. They might be related to the type of diseases treated, but also caused by off-target inhibition as reviewed recently (5).

Beside Btk the homologous kinase Tec is also expressed in platelets. Whereas, FcγRIIa activation and VWF activation of GPIb do not require Tec activation (5, 9), Tec plays a role in GPVI activation. After GPVI-mediated platelet stimulation by high dose collagen, both Btk and Tec support platelet aggregation. Btk-deficient human platelets from patients with XLA and Btk-deficient mouse platelets do not respond to low concentrations of collagen or collagen-related peptide (CRP) indicating that Btk is required for platelet activation after low-degree GPVI stimulation (14, 15). Similar observations have been made by using low Btk-specific concentrations of irreversible BTKi and the reversible BTKi fenebrutinib which inhibits Btk but not Tec and applying human atherosclerotic plaque which also induces only a low-degree activation of GPVI (8, 9, 16–18). After stimulation with high concentrations of collagen, Tec compensates for the absence of Btk (as in XLA patients) or inhibition of Btk (as after platelet pretreatment with Btk-selective concentrations of BTKi) and preserves GPVI-stimulated platelet aggregation. Inhibition of both Tec and Btk abrogates GPVI-activation (15). Since the four approved irreversible covalent BTKi mentioned above have limited selectivity for Btk over Tec as measured by biochemical assays in vitro (5), and at higher concentrations prolong bleeding time in vitro (19), it is assumed that therapeutic concentrations of these BTKi inhibit in platelets irreversibly Tec in addition to Btk thereby abrogating GPVI signaling. This might contribute to the observed bleeding side effects.

Therefore, we hypothesized that off target effects of BTKi with low Btk selectivity over Tec might explain bleeding of BTKi, and investigated in the present study the effects of two novel BTKi on Btk-mediated pathways of platelet aggregation and bleeding time in vitro: the novel selective covalent BTKi remibrutinib (LOU064), a very potent irreversible covalent BTKi, which is highly selective for Btk and barely inhibits Tec (20), and rilzabrutinib (PRN1008) an oral, reversible covalent BTKi which inhibits Btk and Tec with similar IC50 values (21). Both BTKi are in clinical studies of dermatological autoimmune diseases. Rilzabrutinib inhibits very potently Btk and Tec in vitro (IC50 values, 1.3 and 0.8 nM, respectively) (22). It forms a reversible covalent bond with Cys481 of Btk, and shows a fast association and a very slow dissociation rate (23). Rilzabrutinib is in clinical trials of pemphigus (24) and idiopathic thrombocytopenic purpura (ITP), a disease exhibiting very low platelet counts (<50.000/μl) and bleeding events. Here, it inhibits platelet destruction mainly via the inhibition of autoantibody/FcγR signaling in splenic macrophages (25). Unexpectedly, in a previous report clinically relevant concentrations of rilzabrutinib showed no inhibition of platelet activation in vitro (26).

We demonstrate here in our study that (i) remibrutinib and rilzabrutinib inhibit and delay dose-dependently atherosclerotic plaque-induced GPVI-mediated platelet aggregation; (ii) remibrutinib (0.1 μM) and rilzabrutinib (0.5 μM) also block Btk-dependent GPVI-, GPIb/VWF- and FcγRIIa-stimulated platelet aggregation; (iii) higher concentrations of remibrutinib (≥0.2 μM) and therapeutic concentrations of rilzabrutinib (≥0.2 μM) prolong the bleeding time in vitro as measured by PFA-200.

According to the dose-response curve (Figures 1A,B), the potency for platelet inhibition of low degree GPVI-induced platelet aggregation of remibrutinib (IC₅₀ = 0.03 μM) was 5 times higher than that of rilzabrutinib (IC₅₀ = 0.16 μM). Compared with other BTKi (Table 1), remibrutinib is only slightly less potent than fenebrutinib (IC₅₀ = 0.016 μM) and ibrutinib (IC₅₀ = 0.025 μM) and more potent than zanubrutinib, rilzabrutinib, tirabrutinib, acalabrutinib and evobrutinib. The IC₅₀ values of remibrutinib (IC₅₀ = 0.03 μM) and rilzabrutinib (IC₅₀ = 0.16 μM) are 12-times and 2-fold lower than the optimal plasma levels as determined in clinical phase 1 studies, respectively (38, 39). Additionally, both inhibitors induced a dose-dependent increase in delay of atherosclerotic plaque-induced aggregation that was associated with the suppression of aggregation in blood (Figure 1C). A delay was also shown in a previous study using ibrutinib- and acalabrutinib-treated washed platelets stimulated by collagen while the maximal aggregation was unaffected (17).

Remibrutinib (0.1 μM) and rilzabrutinib (0.5 μM) significantly suppressed by >90% GPVI-dependent aggregation on low dose collagen, GPIb/VWF-dependent aggregation on ristocetin stimulation, and FcγRIIA-dependent aggregation upon CD32 cross-linking, but it had no effect on TRAP, AA, or ADP stimulation as expected according to the results of previous studies with other BTKi (7, 9, 16, 17), although it has to be stated that a non-existing effect is difficult to prove and may depend on the experimental conditions.

High dose collagen GPVI-dependent aggregation was suppressed to a similar degree of about 30% with remibrutinib (0.1 μM), rilzabrutinib (0.5 μM) and the Btk-selective reversible BTKi fenebrutinib (0.1 μM) (Supplementary Figure 2). This may indicate that the concentrations and incubation conditions of remibrutinib and rilzabrutinib used are selective for inhibition of Btk and unlikely to also inhibit Tec in platelets. This is unexpected considering the potent inhibition of Tec by rilzabrutinib in vitro (22). Rilzabrutinib by inhibiting Tec in addition to Btk would have shut-off GPVI signaling after high collagen stimulation.

As shown in several studies, low-degree GPVI activation only depends on Btk, while high dose collagen-induced GPVI signaling is also dependent on Tec co-activation (9, 15, 19, 45). In a previous study, 50 nM fenebrutinib was applied and only suppression of platelet aggregation on low but not high dose collagen stimulation was observed (9). Our different results may be explained due to the higher concentration of fenebrutinib (0.1 μM) applied in our study providing a more complete inhibition of Btk.

Our results show that the remibrutinib concentration to fully inhibit Btk-dependent pathways of platelet aggregation (0.1 μM) is lower than the reported maximal plasma level (0.46 μM) in a phase I study after intake of 100 mg q.d. for 12 days (38). Thus, this concentration is expected to block completely Btk-dependent signaling in platelets in vivo. The equivalent rilzabrutinib concentration (0.5 μM) is higher than the plasma C_max reported in clinical studies after therapeutic dosage for autoimmune diseases (0.33 μM) (Table 1) (39). Since the IC₅₀ of rilzabrutinib for inhibition of plaque-induced platelet aggregation in blood was lower (0.16 μM), it is likely that therapeutic concentrations of rilzabrutinib inhibit Btk-dependent pathways of platelet aggregation, but not entirely. Our results are in contrast to findings showing no inhibition of ristocetin- and high dose collagen- induced aggregation of platelet-rich plasma from healthy donors and ITP patients pre-incubated with 1 μM rilzabrutinib for 15 min in vitro (26, 46). The discrepancy might be explained by differences of the experimental system used (blood vs. PRP), different concentrations of collagen (low vs. high) and exposure times of rilzabrutinib (long vs. short). We selected a long exposure time (1 h), since this might better simulate the in vivo situation after absorption of the drug, and previous studies have shown that platelet inhibition with irreversible BTKi increases with the exposure time (17, 19).

Bleeding is a frequent side effect of treatment with certain irreversible BTKi such as ibrutinib and the second generation BTKi acalabrutinib, zanubrutinib, and tirabrutinib used to treat B-cell malignancies (5, 47–49). Exclusive inhibition of Btk should not increase bleeding since XLA patients who are deficient of Btk do not show an impairment of haemostasis (13). It has been discussed that bleeding by these BTKi is related to off-target inhibition of Tec, since this kinase is functionally involved in GPVI-induced platelet activation (8, 15). By comparison, for fenebrutinib, a reversible highly selective BTKi, which is the most selective BTKi and which shows no inhibition of Tec (50), no bleeding events were reported in clinical trials (non-Hodgkin lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, and systemic lupus erythematosus) (5). Also, fenebrutinib in vitro, even at very high concentrations up to 1 μM did not prolong bleeding time measured by the PFA-200 (9).

The reversible BTKi fenebrutinib binds to an inactive conformation of Btk (51). Also, remibrutinib which was developed from fenebrutinib-like scaffolds to bind to the inactive conformation of Btk (20, 51) showed a 175-fold higher affinity for Btk over Tec in binding assays in vitro (20). Thus, it was expected that remibrutinib would not increase in vitro bleeding time measured by PFA-200, similar to fenebrutinib (9). However, we observed that bleeding times in vitro were already slightly but significantly increased after blood incubation with 0.1 μM remibrutinib (which inhibited >90% of Btk-dependent pathways of platelet aggregation), and strongly prolonged by remibrutinib concentrations of 0.2 and 0.5 μM. The results for remibrutinib are similar to a previous study, in which low concentrations of the irreversible BTKi ibrutinib, zanubrutinib, acalabrutinib, and tirabrutinib inhibited GPVI- dependent platelet aggregation by >70%, but 2- to 2.5-fold higher concentrations of these BTKi were required to significantly increase the bleeding time in vitro (19). The increase of closure time was similar to that observed after treatment with low dose aspirin (52).

In a phase I placebo controlled clinical trial of remibrutinib (total 156 healthy subjects), mild self-limited bleeding events were observed only in 4 persons in the multiple-ascending dose cohorts with remibrutinib intake for 12 days. These included two subjects in the 600 mg q.d. cohort with epistaxis and two subjects in the 100 mg cohort with trauma-triggered hematomas (38).

Rilzabrutinib in our study slightly but non-significantly increased at 0.2 μM closure time in the PFA device, the increase was at 0.5 μM pronounced (Figure 4B). Potent Tec inhibition could contribute to the increased in vitro bleeding time (5); however, the results of the aggregation studies upon stimulation with high concentrations of collagen argue against simultaneous Tec inhibition in platelets by 0.5 μM of rilzabrutinib (see above).

Thus, the mechanisms underlying the increase of closure times elicited by remibrutinib as well as rilzabrutinib are unlikely to involve off-target inhibition of Tec. They could be related to effects on the Btk protein itself. Recently it was found that binding of certain irreversible BTKi (except fenebrutinib) to the kinase domain had long-range allosteric effects on the SH2-and SH3- regulatory domains changing their conformation toward an activated state of the protein (53).

In contrast to remibrutinib, there was not a clear difference of rilzabrutinib concentrations that inhibited Btk-dependent pathways of platelet aggregation in the MEA and robustly increased the closure time in the PFA; the concentration of rilzabrutinib of 0.5 μM does both. Maximal therapeutic concentrations of rilzabrutinib (0.33 μM) are expected to significantly increase the closure time in the PFA device, but no treatment-related bleeding had been noted in the ITP clinical trial with rilzabrutinib (25), although the median platelet count at study entry was only 14.173/μl (25). However, 7% (2/27) of patients treated with rilzabrutinib had treatment-related epistaxis as observed in the latest pemphigus clinical trial (24).

Although our in vitro study has the advantage of reducing the complexity of the experimental conditions, and the different effects of the two BTKi studied on platelets in blood are obvious, these data cannot directly be translated into the situation in vivo. Clinical studies of platelet function ex vivo after oral intake of therapeutic dosage are warranted to approach the in vivo effects of remibrutinib and rilzabrutinib on platelets.

In the present study we found significant differences of the two BTKi remibrutinib and rilzabrutinib on platelets that would favor remibrutinib as a candidate for further development as an antiplatelet drug to inhibit Btk-dependent platelet activation pathways underlying atherothrombosis and certain platelet-related immune disorders. Since de novo protein synthesis in platelets is very limited and because low concentrations of irreversible BTKi such as remibrutinib may covalently inactivate platelet BTK already by a single exposure at low concentrations during absorption, it is likely that low doses of such a selective irreversible BTKi are effective in cardiovascular prevention without affecting the immune system (7, 8, 54). Our study further suggests that off-target effects on Tec are unlike to be involved in the increase of closure time measured by PFA, and may not explain the bleeding side effects elicited by BTKi.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by Ethics Committee of the Faculty of Medicine of the University of Munich. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

RD designed and performed experiments, analyzed data, and wrote the manuscript. LG performed experiments and analyzed data. RB provided plaque material. MS supervised experiments and contributed to discussions. CW supervised and contributed to discussions. WS conceived the study, designed experiments, and wrote the manuscript. PH supervised and analyzed experiments and wrote the manuscript. All authors have contributed significantly to this manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft, SFB1123, A2 (PH); RD was sponsored by the Ludwig-Maximilians-University (LMU)-China Scholarship Council (CSC) program.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.