Down Regulation of the Munc18b-syntaxin-11 Complex and β1-tubulin Impairs Secretion and Spreading in Neonatal Platelets

Abstract

Neonatal platelets are hyporeactive and show impaired agonist-induced secretion despite no obvious abnormalities in their granules. Here, we examined, for the first time, the ultrastructure of neonatal and adult platelets following agonist activation. Under resting conditions, neonatal and adult platelets appeared ultrastructurally identical. Following agonist stimulation, however, noticeable degranulation occurred in adult platelets, while granules in neonatal platelets remained clearly visible and apparently unable to centralize or fuse. To investigate the underlying mechanisms, we first examined the expression levels of the main SNARE proteins, which mediate the membrane fusion events required for exocytosis. Neonatal platelets showed significantly reduced levels of syntaxin-11 and its regulator, Munc18b. Since granule centralization depends on contraction of the microtubule ring, we also examined the expression of its main component, β1-tubulin. Noteworthy, we found decreased mRNA and protein levels in neonatal platelets, while and isoforms were overexpressed, partially compensating for that deficiency. Finally, supporting the functional consequences of defective exocytosis, adhesion kinetic assays, performed in plasma-free medium, demonstrated delayed adhesion and spreading of neonatal platelets. This is the first report showing marked reductions of syntaxin-11-Munc18b complex and β1-tubulin in neonatal platelets, indicating that these proteins, required for platelet degranulation, are developmentally regulated.

Tubulin in Platelets: When the Shape Matters

“…Since the centralization of granules is a process that depends on the contraction of the microtubule marginal coil [31,64], and β1-tubulin is its main isoform (up to 90% of total β tubulin in megakaryocytes and platelets), we investigated whether β1-tubulin expression is developmentally regulated. Our findings showed a significant reduction in β1-tubulin mRNA ( TUBB1 ) (~40%) and protein expression levels in neonatal compared to adult platelets (Figure 4A,B with permission) [63]. Neonatal platelets, however, displayed a microtubular ring similar to the present one in adult platelets (Figure 4C,D with permission) [63], suggesting that half the usual amount of β1-tubulin may be sufficient to allow the characteristic discoid shape.…”

“…Our findings showed a significant reduction in β1-tubulin mRNA ( TUBB1 ) (~40%) and protein expression levels in neonatal compared to adult platelets (Figure 4A,B with permission) [63]. Neonatal platelets, however, displayed a microtubular ring similar to the present one in adult platelets (Figure 4C,D with permission) [63], suggesting that half the usual amount of β1-tubulin may be sufficient to allow the characteristic discoid shape. Our results are in agreement with those reported for p.Q43P β1-tubulin variant homozygous carriers, whose platelets, despite having half the normal levels of β1-tubulin, mostly displayed a normal marginal band and a discoid shape [63,65].…”

“…Neonatal platelets, however, displayed a microtubular ring similar to the present one in adult platelets (Figure 4C,D with permission) [63], suggesting that half the usual amount of β1-tubulin may be sufficient to allow the characteristic discoid shape. Our results are in agreement with those reported for p.Q43P β1-tubulin variant homozygous carriers, whose platelets, despite having half the normal levels of β1-tubulin, mostly displayed a normal marginal band and a discoid shape [63,65]. These homozygous PP carriers showed, however, a small percentage of platelets without a clear microtubular ring, but rather they showed a diffuse β1-tubulin in the cytosol [65].…”

“…Despite the absence of differences between neonatal and adult platelets under resting conditions, our group has recently shown that, upon stimulation, adult platelets exhibited dramatic alterations in shape and noticeable degranulation. Neonatal platelets, in contrast, displayed minor shape changes and their granules remained clearly visible throughout the cytoplasm, suggesting a limitation in the granule centering process (Figure 3B) [63].…”

Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia

“…Moreover, the level of P-selectin expression was unaffected following immune thrombocytopenia. Notably, while in humans total P-selectin content is similar between adult and neonatal platelets, 39 in mice it has been suggested that P-selectin expression is developmentally regulated. 40 Thus, the marked reduction in P-selectin secretion induced by CRP and rhodocytin in gestational and early neonatal mice platelets that we have observed may be mediated by a combination of reduced activation of their ITAM receptors and by developmental up-regulation of the α-granule protein.…”