PRT 060318 / AstraZeneca - LARVOL DELTA

PF4 potentiates FcgRIIA stimuli independently of c-Mpl (ISTH 2025) - "Methods We measured aggregation of washed platelets pre-treated with PF4 (10 µg/mL) before addition of the multivalent nanobodies in the absence or presence of inhibitors of JAK2 (ruxolitinib and fedratinib) and Src and Syk kinases (dasatinib and PRT-060318) and a c-Mpl blocking antibody. Nb17-2-Fc but neither Nb17-3 nor PF4 stimulated activation of the NFAT reporter in FcgRIIA-transfected DT40 cells. However, the combination of Nb17-3 and PF4 stimulated NFAT activity."

Hematological Disorders • Thrombocytopenia • SYK

Decreased clot size following stasis and modulated platelet-derived ROS in GPVI deficiency (ISTH 2024) - "Tyrosine kinase inhibitors PRT-060318 (Syk) and dasatinib (Src) significantly reduced ROS in normo-glycaemia conditions (38±8% and 34±19%, respectively, p< 0.05). Clot weight and RBC incorporation were decreased in GPVI-/- versus WT mice (42±11% and 38±16%, respectively, p< 0.05). A significantly reduced thrombus weight after IVC ligation was observed in GPVI-/- vs WT mice (0.87±0.03 and 1.18±0.07, respectively, p< 0.01). No changes in clot firmness were observed following ROTEM analysis."

Cardiovascular • Diabetes • Hematological Disorders • Metabolic Disorders • Thrombosis • SYK

Multi-phased kinetics and interaction of protein kinase signaling in glycoprotein VI-induced platelet αIIbβ3 integrin activation and degranulation. (PubMed, Thromb Haemost) - "Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk and PKC controls platelet granule exocytosis and αIIbβ3 integrin activation."

Journal • MAP2K1 • PLCG2 • SYK

Differential requirement for ligand valency in the activation of CLEC-2 in human and humanised mouse platelets (ISTH 2023) - "We used the Syk inhibitor PRT-060318 to block activation of the kinase... Using FCS and SPR, we show that divalent and higher valency ligands cause clustering of CLEC-2 in cell lines and this leads to an increase in potency. Receptor density and Syk activity were identified as two variables that govern clustering. We show that CLEC-2 is expressed as monomers and dimers in the membrane and that the degree of dimerisation increases with expression thereby increasing the probability of crosslinking CLEC-2 dimers to each other."

Preclinical • SYK

The impact of spleen tyrosine kinase-inhibition on 5B9 HIT antibody-induced procoagulant platelet formation (ISTH 2023) - "5B9 induced a significant formation of procoagulant PLTs (CD62p/PS double positive) in the presence of low- (0.2 IU/mL) dose heparin whereas these changes were absent under buffer conditions or in the presence of high-dose heparin (p=0.0009 and 0.0011, respectively). Formation of Ab-induced procoagulant PLTs seem to be mainly mediated by PLT FcγRIIA as the inhibition of PLT protease activated receptor-1 (PAR-1) with a specific PAR-1 inhibitor did not affect procoagulant PLT formation. Additionally, experiments with the Syk inhibitors Fostamatinib (R406), PRT-060318 and Lanraplenib revealed that procoagulant PLT formation seems to be dependent on Syk-mediated signaling mechanisms as the pretreatment with these inhibitors resulted in a marked reduction of Ab-induced procoagulant PLTs (IC50:14.35±1.27μM; 0.14±1.1μM; and 0.22±1.29μM, respectively).Conclusion(s): Findings of our study suggest that 5B9 HIT Ab induces procoagulant PLTs via the engagement of PLT..."

Cardiovascular • Hematological Disorders • Thrombocytopenia • Thrombosis • FCGR2A • SELP • SYK

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type. (PubMed, Thromb Haemost) - " In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs."

Journal • Cardiovascular • Thrombosis • SYK

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure. (PubMed, J Thromb Haemost) - "Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbβ3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions."

Journal • Thrombosis

Increased platelet procoagulant activity driven by fibrin-GPVI interaction alters clot structure (ISTH 2022) - "Procoagulant platelets were determined, and clot structure analysis assessing clot density, porosity, and retraction was performed in PRP or whole-blood clots from healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib and dasatinib), Affimer proteins inhibiting GPVI-fibrin(ogen) interaction, and eptifibatide control. In the presence of fibrin (and absence of collagen), GPVI-deficient clots, and clots where GPVI-fibrin(ogen) interaction or GPVI-signalling pathway were inhibited (by Affimer and tyrosine kinase inhibitors, respectively) showed fewer procoagulant platelets and decreased fibrin fibre density. Clot porosity was increased in the presence of all inhibitors, however, final clot weight following whole-blood clot retraction was altered only by ibrutinib. The effects of tyrosine kinase inhibitors on procoagulant platelet number were exacerbated by eptifibatide, which also significantly impaired whole-blood clot retraction."

Cardiovascular • Hematological Disorders • Thrombosis

Fully automated platelet Differential Interference Contrast image analysis via deep learning. (ISTH 2022) - "Platelets were spread in the presence and absence of inhibitors (dasatinib, ibrutinib, and PRT-060318) or agonist (thrombin) known to impact platelet morphology, and over a range of different substrates (CRP-XL, vWF and fibrinogen). A convolutional neural network (CNN) allowed fully automated analysis of platelet spreading assays captured by DIC microscopy when platelets were spread over a range of substrates, and when in the presence or absence of inhibitors or agonist. Significant variation was observed between individual annotators, highlighting biases associated with manual analyses. Results were consistent in quantifying the spread area of platelets, which were comparable to published literature."

Cardiovascular • Hematological Disorders • Myocardial Infarction • Thrombosis

Investigations to assess Impact of Syk-inhibition on antibody-mediated desialylation: novel implications on therapy for immune thrombocytopenia (ISTH 2022) - "Our data shows that Syk inhibition can effectively alter antibody-mediated biological effects in ITP. Activation of FcyRIIA via crosslinking resulted in significant increase in apoptosis and desialylation markers. R406 and PRT318 significantly inhibited apoptosis (mean %PS externalization ±SEM: 27.9±2.1 vs."

Hematological Disorders • Thrombocytopenia • Thrombocytopenic Purpura • FCGR2A • FCGR2B • SYK

Spleen Tyrosine Kinase Activation Drives Platelet Procoagulation in Antiphospholipid Syndrome During Pregnancy (ISTH 2022) - "Background: Pregnant patients with antiphospholipid syndrome (APS) develop thrombotic and obstetric complications despite therapy with low-molecular-weight heparin (LMWH) and aspirin...Treatment of APS/SLE or PC+ platelets with the Syk inhibitor PRT-060318 (10 µM) significantly attenuated Syk phosphorylation, P-selectin expression, and PS exposure... Circulating ballooned platelets typified all three trimesters of pregnancy in APS/SLE participants. Platelet activation and P-selectin expression increased as pregnancy progressed and peaked during gestational week 24-28. Expectedly, phosphatidylserine (PS) externalisation (indicated by platelet membrane annexin-V binding) and thrombin generation followed a similar temporal pattern."

Cardiovascular • Genetic Disorders • Hematological Disorders • Immunology • Inflammatory Arthritis • Lupus • Obstetrics • Systemic Lupus Erythematosus • Thrombosis • ANXA5 • SYK

Fully automated platelet differential interference contrast image analysis via deep learning. (PubMed, Sci Rep) - "The CNN effectively analysed platelet morphology when platelets spread over a range of substrates (CRP-XL, vWF and fibrinogen), in the presence and absence of inhibitors (dasatinib, ibrutinib and PRT-060318) and agonist (thrombin), with results consistent in quantifying spread platelet area which is comparable to published literature. The application of a CNN enables, for the first time, automated analysis of platelet spreading assays captured by DIC microscopy."

Journal • Cardiovascular • Hematological Disorders • Myocardial Infarction • Thrombosis

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear. (PubMed, Int J Mol Sci) - "We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment...A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A (TxA), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen."

Journal • Atherosclerosis • Cardiovascular • Dyslipidemia • Hematological Disorders • Thrombosis • SYK

Novel multimeric anti-GPVI nanobody and modelling reveals mechanism of GPVI clustering and activation (ECTH 2021) - "Tetrameric NB2 (16 nM) induced GPVI-mediated platelet aggregation, which was inhibited by Src and Syk kinase inhibitor PP2 (20 pM) and PRT-060318 (10 pM), and GPVI inhibitor JAQ1-Fab (200 nM) and monomeric NB2 (100 nM)... These findings indicate that monomeric NB2 and dimeric NB2 are GPVI inhibitors while tetrameric NB2 is a GPVI agonist. The results show that dimerisation of GPVI does not induce activation of human platelets, in contrast to results on mouse platelets, and higher order multimers are required for activation. The study is funded by the European Union's Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant agreement No."

SYK

The Serine/Threonine Protein Phosphatase 2A (PP2A) Regulates Syk Activity in Human Platelets. (PubMed, Int J Mol Sci) - "Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation."

Journal • SYK

[VIRTUAL] Non-redundant Roles of Platelet Glycoprotein VI and IIb/IIIa via Syk Kinase in Fibrin-dependent Thrombus Shielding under Flow (ISTH 2020) - "With or without FXIIIa, fibrin surfaces moderately support thrombus formation, when compared to collagens. Formation of the shielding thrombus relies on GPIIb/IIIa and Syk activation, and on essential but low GPVI activity. These results indicate that fibrin triggers a low-level activation of GPVI, which is dependent on GPIIb/IIIa for inducing Syk-mediated platelet activation."

Hematological Disorders • Thrombosis • F2 • Factor XIIIa • SYK

Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces. (PubMed, Int J Mol Sci) - "Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO) sequence."

Journal