Search

The hallmark of haemophilia A (HA) therapy is prophylaxis, aimed at spontaneous bleeding prevention. Emicizumab provides a viable alternative to intravenous factor replacement therapy. However, data on its use in infants are limited. This single-centre open arm prospective study reports on emicizumab prophylaxis in infants. We included severe HA patients under 1 year who started emicizumab prophylaxis since 2018, with longitudinal follow-up. The study collected data on demographics, clinical and laboratory variables, the occurrence of bleeding events, surgeries and treatment outcomes. Of the 27 enrolled infants, whose median age at prophylaxis initiation was 7 months, 24 primarily choose to start emicizumab therapy (3/27 switched from FVIII prophylaxis due to development of FVIII inhibitors). The median age for prophylaxis initiation decreased to 3 months in 2023. Following emicizumab initiation, the median calculated ABR decreased, and no intracranial haemorrhages were observed. Thrombin generation showed a significant improvement in peak height and endogenous thrombin potential at steady state after a loading period. Our study highlights a shift towards early prophylaxis in the era of non-replacement therapies. It underscores the need for continuous evaluation and refinement of treatment approaches, emphasizing personalized care and diligent monitoring in the evolving field of paediatric haemophilia care., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Unlabelled: The action of clopidogrel on platelet receptors was analysed using platelets obtained from 11 healthy volunteers given 75 mg of clopidogrel daily for 8 d. Samples of blood were taken before treatment and after 8 d of medication. Determination of 2-methylthioadenosine diphosphate trisodium (2MesADP)-induced platelet aggregation, serine/threonine and tyrosine phosphorylations were performed in the absence or presence of the P2Y1-receptor-specific antagonist: adenosine 3'-phosphate 5'-phosphate (A3P5P) or the strong inhibitor of GPIIb/IIIa activation: SR121566., Major Conclusions: 1). Serine and threonine phosphorylations of the myosin light chain (P20) and pleckstrin (P47) do not behave similarly, although they are both recognized as the result of phospholipase C pathway stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphorylation occurs in the presence of A3P5P, suggesting that the P2Y12 receptor signal contributes to P20 phosphorylation mediated by a calcium-independent pathway. The results suggest that P2Y1 and P2Y12 receptors interact to modulate the phosphorylation of P20 and P47. 2). The inside-out signalling dependent on both P2Y12 and P2Y1 is necessary for GPIIb/IIIa activation. 3). Clopidogrel and SR121566 inhibited the increase in tyrosine phosphorylation induced by 2MesADP and concomitantly inhibited platelet aggregation, indicating that most of the phosphorylations are GPIIb/IIIa dependent. However, neither clopidogrel nor SR121566 inhibited the first wave of 80 kDa substrate (cortactin) which is involved in the reorganization of the cytoskeleton necessary for shape change and which appeared to be essentially P2Y1 dependent.

Increased Ca2+ signal generation may lead to hyperactivity of platelets and contribute to thrombotic complications. Using fura-2-loaded platelets from 51 healthy volunteers, high variability was detected in the Ca2+ responses evoked by the receptor agonists, thrombin and collagen, and the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), thapsigargin (Tg). Oral intake of 500mg aspirin reduced the magnitude of the Ca2+ responses, and lowered the intra-individual coefficients of variance of the responses by 50%. However, the corresponding inter-individual variance coefficients were only a little influenced by aspirin intake, pointing to subject-dependent factors in Ca2+ handling that are unrelated to thromboxane formation. With each agonist, 6-9% of the subjects had platelets with relatively high Ca2+ responses (> mean + SD) both before and after aspirin intake. In 90% (9/10) of these cases the high responsiveness was confirmed in platelets obtained 6-12 months later. The Tg- but not thrombin-induced Ca2+ responses correlated inversely with the expression levels of SERCA PL/IM 430 (SERCA-3b) in platelets. After aspirin intake, the Ca2+ responses with collagen but not thrombin correlated inversely with SERCA-2b expression. These results suggest that, in the absence of potentiating effects of thromboxane, (i) the amount of PL/IM 430-recognizable SERCA may control the Ca2+ signal when SERCA-2b is specifically inhibited (with Tg), and (ii) the expression of SERCA-2b determine the collagen- but not the thrombin-evoked Ca2+ signal. Accordingly, limited Ca2+-pumping activity by low expression of one of the SERCA isoforms is likely to be one of the factors resulting in increased platelet activity towards collagen or thapsigargin but not thrombin.

It is unknown whether bone changes which can occur in multiple myeloma (MM) are due to cytokine-induced osteoclastic bone resorption from a clone of abnormal plasma cells or high-dose glucocorticoid therapy. We studied 25 MM patients treated for 1-12 years with combination chemotherapy, subdivided into two groups. Group 1 consisted of 12 patients with stage I and II myeloma and group 2 consisted of 13 patients with stage III MM. Their serum biochemistry, tetracycline-labelled bone histomorphometry and bone densitometry were compared to age- and sex-matched controls. Patients with MM demonstrated increased indices of bone resorption (P < 0.001 versus controls) and, to a lesser extent, increased indices of bone formation (P < 0.01 versus controls). No patient had evidence of a mineralization defect. Lumbar spine, femoral neck and total body bone mineral density measurements (BMD) were significantly lower in group 2 compared with group 1 (P < 0.05). Following 12 months of therapy, lumbar spine BMD decreased by 6.6% (95% CI, 2.7% to -9.3%) and femoral neck BMD decreased by 9.5% (95% CI, -3.2% to -15.9%). In a stepwise regression analysis, cumulative prednisolone dosage (B Coef. = -0.39; P = 0.03) and plasma cell infiltrate (B Coef. = -0.08; P = 0.05) were the most important predictors of lumbar spine bone loss, whereas serum paraprotein (B Coef.= -0.35; P = 0.02) and plasma cell infiltrate (B Coef. = -0.20; P = 0.04) were the most important predictors of femoral neck bone loss. We conclude that MM is characterized by high bone turnover with osteoblast-osteoclast uncoupling. Both disease activity and high-dose glucocorticoid therapy may be responsible for the ongoing bone loss seen with MM.

Phosphorylations induced by 2-MeS-ADP, a potent agonist of platelet ADP receptors, have been studied in rat platelets, and the effect of clopidogrel, a compound which inhibits platelet aggregation by selectively reducing the binding of ADP to its low affinity receptors on platelets, has been determined. 2-MeS-ADP induced platelet activation (shape change and aggregation) simultaneously with the phosphorylation of myosin light chain (P20) and plekstrin (P47). Phosphorylation of P20 and P47 was transient, a maximum being observed 10 s after addition of the agonist when shape change reached its maximum. P20 and P47 phosphorylations were not strongly affected by clopidogrel treatment. Following stimulation of platelets with 2-MeS-ADP, several proteins were phosphorylated at tyrosine residues. Clopidogrel treatment inhibited the increase in phosphorylation of P140, P100, P80/85, P66 and P55 concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P80/85 kD doublet which occurs at the time of the shape change. P80/85, identified by immunodetection as cortactin, could be involved in the reorganization of the cytoskeleton necessary for morphological changes. Thus, by using clopidogrel-treated rat platelets, we were able to determine some of the phosphorylations coupled either to clopidogrel-resistant high-affinity ADP receptors leading to shape change or to clopidogrel sensitive low-affinity ADP receptors coupled to the aggregation process.

Several functions have been proposed for Rap1B in human platelets, including the regulation of phospholipase (PL) C gamma and Ca2+ ATPase. However, its localization is largely unknown. In the present study we have investigated the subcellular distribution of Rap1 by immunocytochemical techniques using affinity purified polyclonal antibodies raised against residues 121-137 common to the 95% homologous Rap1A and Rap1B proteins. By immunofluorescence, a positive labelling was obtained on intact resting platelets and was abolished after adsorption of the antibodies with the control peptide. Immunoelectron microscopy was then used to further define the subcellular localization of Rap1B in platelets and megakaryocytes (MK). In resting cells, immunolabelling for Rap1B was associated with the plasma membrane, mostly at its inner face, and lined the membrane of the open canalicular system (OCS). Some labelling was also found outlining the alpha-granules, identified as such by a double labelling with an anti-GPIIb-IIIa. On thrombasthenic platelets the same localization was observed. When platelets were stimulated by thrombin, immunolabelling for Rap1B was redistributed to the zones of fusion of the granules with the OCS, and to the plasma membrane with a higher concentration on pseudopods. Human MK expressed Rap1 and the staining revealed the association of the protein with the demarcation membranes and alpha-granules. This study presents a first approach to the localization of a small GTP binding-protein Rap1B in whole platelets and MK, and shows its association with both the plasma and OCS membranes, as well as with the alpha-granule membranes.

The responses to alpha- and gamma-thrombin were studied in normal and Bernard-Soulier platelets labelled with [32P]phosphate, to investigate the relationship between thrombin binding to the platelet membrane glycoprotein Ib (GPIb) and thrombin-induced platelet activation. For this purpose we conducted parallel studies of the kinetics of platelet aggregation, granule secretion, hydrolysis of polyphosphoinositides, formation of phosphatidic acid, phosphorylation of the myosin light chain (p20) and of the 43 kDa protein (p43), and thromboxane B2 formation. Like alpha-thrombin, gamma-thrombin activated control platelets via all the above metabolic responses, but only after a prolonged lag. In Bernard-Soulier platelets, alpha-thrombin induced polyphosphoinositide hydrolysis and phosphatidic acid formation, p20 and p43 phosphorylation, thromboxane B2 formation, secretion and to a lesser extent aggregation, but only after a prolonged lag. The metabolic responses of Bernard-Soulier platelets to gamma-thrombin were very similar to those of control platelets. We have previously showed that GPIb which is not present in Bernard-Soulier platelets binds alpha- but not gamma-thrombin. The present results indicate that thrombin binding to GPIb is not directly coupled either with the activation of phospholipase C specific to polyphosphoinositides, or with the activation of protein kinase C and phospholipase A2. However, thrombin binding to GPIb appears to promote an early mechanism which accelerates all the platelet responses.

A possible relationship between binding sites for Immunoglobulin E (IgE) on human platelets, involved in IgE-dependent cytotoxic functions of platelets against helminth parasites, and well-characterized platelet constituents involved in haemostasis, was investigated. We first explored the interaction with IgE of platelets from patients with rare inherited deficiencies of defined platelet constituents and functions: Glanzmann's thrombasthenia, Bernard-Soulier and grey platelet syndromes. We report that only type I and II thrombasthenic platelets, which lack the membrane glycoproteins (GP) IIb and IIIa, failed to bind IgE and to exhibit IgE-dependent effector functions. Since thrombasthenic monocytes, however, showed normal interaction with IgE, this defect appeared restricted to platelets. Polyclonal and monoclonal antibodies directed against GP IIb-IIIa complex, but not monoclonal antibody directed against GP Ib, inhibited the binding of IgE to normal platelets, and their IgE-dependent cytotoxicity. Taken together, these findings indicate a relation between the GP IIb-IIIa complex and the expression of IgE binding sites and IgE-dependent effector functions in human platelets.

Calcium uptake into isolated membrane vesicles from two patients with a grey platelet syndrome has been investigated. An increase in calcium transport appears in both patients when compared to controls. Determination of the kinetic parameters of the calcium transport system gave similar apparent affinity for calcium and an increase in the calcium uptake velocity. This increase in calcium transport is correlated with the increase of the associated Ca2+ activated ATPase activity. The results would suggest a new relationship between the ultrastructural and functional abnormalities of the grey platelet syndrome.

Prothrombinase activities of platelets have been measured in diluted platelet-rich plasma using a chromogenic substrate assay and purified coagulation factors. No abnormalities in prothrombinase activities were found for platelets from patients with storage pool disease (dense-body deficiency), grey platelet syndrome, and Glanzmann's thrombasthenia. It is concluded that neither release of dense bodies and alpha-granules nor aggregation of platelets are essential prerequisites for exposure of a procoagulant surface. Platelets from patients with Bernard-Soulier syndrome, however, have approximately 10-fold higher prothrombinase activities in the non-stimulated form than normal non-stimulated platelets. The increased procoagulant activity cannot be completely ascribed to an increase in platelet size. It is suggested that the increased prothrombinase activity reflects an increased exposure of phosphatidylserine at the outer surface of non-stimulated Bernard-Soulier platelets, earlier described by Perret et al (1983).

Washed platelets isolated from one patient suffering from the inherited grey platelet syndrome were studied during thrombin-induced activation. The agonist-induced changes in (i) morphology, (ii) typical functional cell responses, (iii) membrane phospholipid metabolism and protein phosphorylation were studied and compared with the changes obtained with normal platelets. The morphology of the platelets as visualized by electron microscopy confirmed the almost total absence of intracellular alpha-granules and marked vacuolization. During thrombin stimulation the morphological changes were clearly delayed as compared to normal platelets, the granule centralization and aggregation occurred only 15 s after thrombin addition instead of 5 s in normal platelets. After 15 s, however, even though no alpha-granules were observed, a ring-like structure occurred centrally, indicating that they are not a prerequisite for this reaction. The whole release reaction, i.e. liberation of [14C]serotonin from dense granules and beta-N-acetylglucosaminidase activity from lysosomes, and the thromboxane synthesis were delayed and remained lower than in normal platelets. No thrombin-induced phosphatidyl 4,5-bisphosphate breakdown was measurable on 32P-prelabelled platelets although [32P]phosphatidate formation occurred normally. Phosphorylation time courses of myosin light chain (P20) and of protein P43 (mol wt 43,000) markedly differed from those of controls, being less than half of the normal during the first 15 s and remaining subnormal even after complete aggregation. These results suggest that in platelets devoid of alpha-granules a deficient transmembrane signalling system is likely responsible for the impaired physiological responses.

An IgG antiplatelet antibody found in a multitransfused patient with Bernard-Soulier syndrome (BSS), reacted with a normal platelet surface antigen of 150 000 daltons which was similar to the glycoprotein missing from BSS platelets. The BSS platelet antibody (BSS-Pab) aggregated all control platelets which then released ADP and 5-HT and synthesized thromboxane. When mixed with the antibody, BSS platelets did not aggregate, did not release ADP and 5-HT and failed to synthesize thromboxane. The BSS-Pab was not inactivated by incubation with BSS platelet stroma. While the antibody did not aggregate thrombasthenic platelets, its aggregating activity was lost after incubation with their stroma. The BSS-Pab did not provoke ADP or 5-HT release or thromboxane synthesis in thrombasthenic platelets or in the platelets of a patient with platelet cyclooxygenase deficiency or in normal platelets treated with indomethacin. The aggregating, release and synthetic responses of platelets after binding of BSS-Pab to its membrane antigen (probably glycoprotein I) requires the presence of glycoprotein IIb and/or IIa and the normal metabolism of arachidonic acid.