Bruton tyrosine kinase (Btk) is expressed in B-lymphocytes, myeloid cells and platelets, and Btk-inhibitors (BTKi) are used to treat patients with B-cell malignancies, developed against autoimmune ...diseases, have been proposed as novel antithrombotic drugs, and been tested in patients with severe COVID-19. However, mild bleeding is frequent in patients with B-cell malignancies treated with the irreversible BTKi ibrutinib and the recently approved 2nd generation BTKi acalabrutinib, zanubrutinib and tirabrutinib, and also in volunteers receiving in a phase-1 study the novel irreversible BTKi BI-705564. In contrast, no bleeding has been reported in clinical trials of other BTKi. These include the brain-penetrant irreversible tolebrutinib and evobrutinib (against multiple sclerosis), the irreversible branebrutinib, the reversible BMS-986142 and fenebrutinib (targeting rheumatoid arthritis and lupus erythematodes), and the reversible covalent rilzabrutinib (against pemphigus and immune thrombocytopenia). Remibrutinib, a novel highly selective covalent BTKi, is currently in clinical studies of autoimmune dermatological disorders. This review describes twelve BTKi approved or in clinical trials. By focusing on their pharmacological properties, targeted disease, bleeding side effects and actions on platelets it attempts to clarify the mechanisms underlying bleeding. Specific platelet function tests in blood might help to estimate the probability of bleeding of newly developed BTKi.
Lysophosphatidic acid (LPA) is a potent bioactive phospholipid. As many other biological active lipids, LPA is an autacoid: it is formed locally on demand, and it acts locally near its site of ...synthesis. LPA has a plethora of biological activities on blood cells (platelets, monocytes) and cells of the vessel wall (endothelial cells, smooth muscle cells, macrophages) that are all key players in atherosclerotic and atherothrombotic processes. The specific cellular actions of LPA are determined by its multifaceted molecular structures, the expression of multiple G‐protein coupled LPA receptors at the cell surface and their diverse coupling to intracellular signalling pathways. Numerous studies have now shown that LPA has thrombogenic and atherogenic actions. Here, we aim to provide a comprehensive, yet concise, thoughtful and critical review of this exciting research area and to pinpoint potential pharmacological targets for inhibiting thrombogenic and atherogenic activities of LPA. We hope that the review will serve to accelerate knowledge of basic and clinical science, and to foster drug development in the field of LPA and atherosclerotic/atherothrombotic diseases.
Anti-platelet drugs reduce arterial thrombosis after plaque rupture and erosion, prevent stent thrombosis and are used to prevent and treat myocardial infarction and ischaemic stroke. Some of them ...may also be helpful in treating less frequent diseases such as thrombotic thrombocytopenic purpura. The present concise review aims to cover current and future developments of anti-platelet drugs interfering with the interaction of von Willebrand factor (VWF) with glycoprotein (GP) Ibα, and directed against GPVI, GPIIb/IIIa (integrin α
β
), the thrombin receptor PAR-1, and the ADP receptor P2Y
. The high expectations of having novel antiplatelet drugs which selectively inhibit arterial thrombosis without interfering with normal haemostasis could possibly be met in the near future.
Several methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry ...(MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p < 0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.
Objectives The aim of this study was to understand the initial mechanism of arterial thrombus formation induced by vulnerable human atherosclerotic plaques to re-assess and improve current ...antithrombotic strategies. Background Rupture of atherosclerotic plaques causes arterial thrombus formation that might lead to myocardial infarction and ischemic stroke. Atherothrombosis is considered as an inseparable tangle of platelet activation and coagulation processes, involving plaque components such as tissue factor (TF) and collagen as well as blood-borne TF and coagulation factor XIIa (FXIIa). A combination of anticoagulants and antiplatelet agents is the present treatment. Methods Human atheromatous plaque material was exposed to blood or blood components at physiological calcium/magnesium concentration. Platelet aggregation and coagulation were measured under static and arterial flow conditions by state-of-the-art microscopic and physiological techniques. Plaque TF, plaque collagen, FXIIa, and platelet glycoprotein VI (GPVI) were specifically inhibited. Results Plaques induced thrombus formation by 2 discrete steps. The rapid first phase of GPVI-mediated platelet adhesion and aggregation onto plaque collagen occurred within 1 min. The second phase of coagulation started after a delay of >3 min with the formation of thrombin and fibrin, and was driven entirely by plaque TF. Coagulation occurred only in flow niches provided by platelet aggregates, with no evidence for a role of blood-borne TF and FXIIa. Inhibition of GPVI but not plaque TF inhibited plaque-induced thrombus formation. Conclusions The major thrombogenic plaque components—collagen and TF—induce platelet activation and coagulation, respectively, in 2 consecutive steps. Targeting specifically the first step is crucial and might be sufficient to inhibit atherothrombus formation.
High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in ...stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2-3 min. In our cohort of healthy blood donors (
= 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.
Lysophosphatidic acid (LPA) is a ligand for LPA1–3 of the endothelial differentiation gene family G-protein-coupled receptors, and LPA4–8 is related to the purinergic family G-protein-coupled ...receptor. Because the structure-activity relationship (SAR) of GPR92/LPA5 is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA5 residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA5 were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA5 using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates ≫ N-arachidonoylglycine. These results confirm LPA5 to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA5 agonists. A pharmacophore model of LPA5 binding requirements was developed for in silico screening, which identified two non-lipid LPA5 antagonists. Because LPA5 transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA5 antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA5 may provide a basis for future anti-thrombotic therapies.
Migration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase ...inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1) phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood.
In this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1) to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1) to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold) in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a) leading to G(1) arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays.
The results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear actin might be critical in the regulation of these fundamental endothelial functions.
PDF
