Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

Purpose Cyclin‐dependent kinase (CDK) 4/6 inhibitors are integral treatment for advanced hormone receptor positive breast cancer; however, venous thromboembolic events (VTE) occurred in 1%‐5% of clinical trial patients. Thrombosis rates in the real‐world setting remain unclear. We aimed to define the rate of thromboembolic events, risk factors for thrombosis on CDK 4/6 inhibitors and evaluate the Khorana VTE risk score as a predictive tool for VTE in patients on CDK 4/6 therapy. Methods Multicenter retrospective analysis of adult breast cancer patients prescribed palbociclib, ribociclib, or abemaciclib. The primary endpoint was thrombosis during treatment or within 30 days of CDK inhibitor discontinuation. Cox regression was used to model time‐to‐thrombosis, starting from a patient's initiation of CDK 4/6 therapy. The extended Kaplan‐Meier method and Cox modeling were used to assess the effect of time‐varying thrombosis status on overall survival. Results Two hundred and sixty‐six patients were included (89% on palbociclib, 14% on abemaciclib, 7% on ribociclib). Twenty‐nine thrombotic events occurred in 26 (9.8%) women. Of these events, 72% were venous and 34% were arterial. The 1‐year incidence of thrombosis was 10.4% overall, 10.9% on palbociclib, 8.3% on ribociclib, and 4.8% on abemaciclib. Hemoglobin less than 10 g/dL was a statistically significant predictor of thrombosis (HR 3.53, P: .014). Khorana score ranged from 0‐3, with the majority between 0 and 1 and was not predictive of VTE. Thrombosis was associated with reduced overall survival (HR 1.28, P: .128, median 7.3 months) compared to not having a CDK‐associated clot (median 35.7 months). Discussion VTE in our analysis is higher than reported in clinical trials and arterial thrombosis comprised over one‐third of events. The highest incidence was with palbociclib, followed by ribociclib. Khorana score did not predict VTE risk. Larger, real‐world studies are needed. The role for prophylactic anticoagulation is yet to be defined in this patient population.

Activation of coagulation factor (F) XI promotes multi-organ failure in rodent models of sepsis and in a baboon model of lethal systemic inflammation induced by infusion of heat-inactivated Staphylococcus (S.) aureus. Here we employed the anticoagulant FXII-neutralizing antibody 5C12 to verify the mechanistic role of FXII in this baboon model. Compared to untreated controls, repeated 5C12 administration before and at 8h and 24h after bacterial challenge prevented the dramatic increase in circulating complexes of contact system enzymes FXIIa, FXIa and kallikrein with antithrombin or C1 inhibitor, and prevented cleavage and consumption of high-molecular-weight kininogen. Activation of several coagulation factors and fibrinolytic enzymes were also prevented. D-dimer levels showed profound increase in untreated but not in the treated animals. The antibody also blocked the increase in plasma biomarkers of inflammation and cell damage, including tumor necrosis factor, interleukin (IL)-1β, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, nucleosomes, and myeloperoxidase. Based on clinical presentation and circulating biomarkers, inhibition of FXII prevented fever, terminal hypotension, respiratory distress and multi-organ failure. All animals receiving 5C12 had milder and transient clinical symptoms and were asymptomatic at day 7, while untreated controls suffered irreversible multi-organ failure and had to be euthanized within 2 days after the bacterial challenge. This study confirms and extends our previous finding that at least two enzymes of the contact activation complex, FXIa and FXIIa, play critical roles in the development of an acute and terminal inflammatory response in baboons challenged with heat-inactivated S. aureus.

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