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New antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque-mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non-Btk-mediated effects. Btk-deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy.To compare the antithrombotic effects of the highly selective Btk inhibitor AB-95-LH34 (LH34) with ibrutinib.Glycoprotein VI and G-protein coupled receptor-mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi-aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro.LH34 showed similar inhibition of Btk-Y223 phosphorylation as ibrutinib, but had no off-target inhibition of Src-Y418 phosphorylation. Similar dose-dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2β1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P-selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque-induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib.LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and procoagulant platelet activity in vitro, with less off-target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects.

Acute thrombotic thrombocytopenic purpura (TTP) is a life-threatening emergency and plasma exchange (PEX) is the initial treatment shown to reduce acute mortality.To compare current practice in the United Kingdom (UK) against the standards set out in the 2012 British Society of Haematology guideline, and to better understand the issues affecting prompt initiation of PEX.The trainee research network HaemSTAR conducted a retrospective nationwide review of adults presenting to UK hospitals with a first episode of acute TTP.Data on 148 patients treated at 80 UK hospitals between 2014 and 2019 demonstrated that 64.8% of patients received PEX within 24 h. Diagnostic uncertainty was the most commonly cited reason for delayed treatment. Conversely, a shorter time to PEX occurred in patients who had red cell fragments or severe thrombocytopenia identified on their first complete blood count. Availability of on-site PEX was associated with a greater proportion of patients receiving PEX within 8 h compared to patients transferred, but by 24 h there was no difference between the two groups and two-thirds of all patients had received their first PEX. The mortality rate for patients that received PEX was 9.2%, with 27.8% of deaths linked to delayed treatment initiation.This is the first multi-center evaluation of treatment delays in acute TTP and it will inform targeted pathways to improve prompt access to life-saving intervention.

Anti-platelet factor 4 (PF4) antibodies that activate platelets via FcγRIIA drive the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT). Evolution of these antibodies and their ability to activate platelets after initial treatment remains unknown.To assess how clinical and platelet parameters, anti-PF4 antibody levels, and patient serum reactivity changes during follow-up after VITT presentation.We describe cases of seven discharged VITT patients that were followed from diagnosis up to 280 days (range 199-280) after vaccination. We measured anti-PF4 antibodies and PF4 levels in patient serum during follow-up and tested the ability of patient serum to activate healthy donor platelets and patient platelets over time.Anti-PF4 immunoglobulin G antibody levels are very high at diagnosis (0.9-2.6 OD) and remain relatively high (1.0 OD) in all patients, except one treated with rituximab, at 7 months post vaccination. All patients were on direct oral anticoagulants throughout follow-up and no patients had recurrent thrombosis. Patients' platelets during follow-up have normal FcγRIIA levels and responsiveness to platelet agonists. Patient diagnostic serum strongly activated control platelets, either alone or with PF4. Most follow-up serum alone was weaker at stimulating donor and patient platelets. However, follow-up serum beyond 150 days still strongly activated platelets with PF4 addition in three patients. Patient serum PF4 levels were lower than controls at diagnosis but returned within normal range by day 50.Explanations for reduced platelet activation during follow-up, despite similar total anti-PF4 antibody levels, remains unclear. Clinical implications of persistent anti-PF4 antibodies in VITT require further study.

Microtubules are ubiquitously expressed cytoskeletal structures responsible for a host of cellular processes from division to cargo transport. In specialised cells, the expression of specific isoforms of tubulin and their subsequent post-translational modifications are thought to drive and co-ordinate unique morphologies and behaviours. The mechanisms by which {beta}-1 tubulin (encoded by TUBB1), the platelet and megakaryocytic specific {beta}-tubulin isoform required for platelet production and function, drives these processes remains poorly understood. We investigate the effects of two key tubulin post-translational polymodifications (polyglutamylation and polyglycylation) on the glutamate rich C-terminus of {beta}-1 tubulin using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived megakaryocytes, and healthy human donor platelets. We find that while megakaryocytes (MKs) are positive for both polymodifications, polyglycylation is substantially reduced on platelets. On platelet activation, the marginal band becomes heavily polyglutamylated, which drives the mobilisation of motor proteins, including axonemal dynein, to achieve the shape change required for the haemostatic role of platelets. Finally, we show that a number of modifying enzymes (Tubulin Tyrosine Like Ligases (TTLLs) and Cytosolic Carboxypeptidases (CCPs)) are up-regulated through MK maturation. In platelets, a single polyglutamylase (TTLL7) is expressed to mediate the polyglutamylation of the marginal band required for shape change on activation. Finally, we report a novel disease causing gene in multiple families (TTLL10) resulting in bleeding despite normal platelet production and function. This work highlights the importance of a complex regulatory mechanism driven by both cell specific tubulin isoform expression and differential post-translational modification to drive specialist function, the loss of which results in disease states.nnnnO_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/595868_fig1.gif" ALT="Figure 1">nView larger version (20K):norg.highwire.dtl.DTLVardef@2ea097org.highwire.dtl.DTLVardef@131582corg.highwire.dtl.DTLVardef@93b671org.highwire.dtl.DTLVardef@1aee018_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Graphical AbstractIn this work we report a system of reversible polymodification (polyglutamylation and polyglycylation) of the MK and platelet specific -1 tubulin isoform required for platelet production and function. These polymodifications are driven by an up-regulation of key Tubulin Tyrosine Like Ligases (TTLLs) which mediate the addition of single glutamate or glycine residues (initiases which trigger monogluytamylation or monoglycylation), and elongases which extend a glutamate or glycine tail (polyglutamylation and polyglycylation respectively). These processes are reversed by Cytosolic Carboxypeptidase (CCP) enzymes). MKs express a range of TTLLs and CCPs, while platelets only express a single polyglutamylase, TTLL7.nnC_FIG