Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders characterized by impairments in reciprocal social interactions, verbal and non-verbal communication, and stereotyped behavior. The prevalence of ASD is estimated to be around 1.5 % of the population and has risen rapidly the past decade making it now one of the most prevalent disorders in childhood [
1]. Although ASD is a highly heritable disorder [
2], the genetic cause is complex and involves a multitude of genes, genetic heterogeneity, imprinting as well as gene-environment interactions [
3‐
5]. Gene pathway analysis studies have provided insight in the diverse nature of the genetic abnormalities, and it now becomes evident that the majority of genetic defects influence a limited number of core functional mechanisms in the brain [
6‐
10]. These identified networks are mainly related to the neuronal synapse and include synaptic plasticity, structure, signaling, and transcriptional regulation. Studying synaptic function in vivo and modeling the effect of single gene mutations in vitro are challenging despite the availability of non-invasive technologies to study human brain function and the development of several animal models [
11‐
13].
Blood platelets and neurons that seem to share some typical characteristics as subclinical platelet abnormalities were often detected in monogenetic and complex neurological diseases [
14,
15]. Detailed functional platelet testing was performed to gain insights in the disease mechanisms of some monogenetic neuropathologies by elucidating abnormalities in transcription factors, membrane transporters, G-protein signal transduction, and cytoskeletal proteins (e.g., Rett syndrome [
16], intellectual disability, and others [
17‐
19]). However, in the early 1970s, studies focused mainly only on the overlapping serotonin metabolism in neurons and platelets [
20,
21], both comprising the active transport system for serotonin [
22], storage into specialized dense core granules, and availability of monoamine oxidase in mitochondria. Since then, additional research has shown that both neurons and platelets contain overlapping gene expression profiles, similar types of granules loaded with partially the same signaling molecules such as ATP and serotonin, and that both cell types use Ca
2+, intracellular inositol phosphate and the prostaglandin pathway as initiators of granule secretion. Moreover, the highly specialized process of regulated granule trafficking and secretion found in neurons mirrors at least partially that of platelets [
23,
24]. Abnormalities in the platelet serotonin metabolism have been described in different complex neurological diseases, including schizophrenia [
25], migraine [
26], and Alzheimer’s disease [
27]. Elevated blood serotonin level is also the first quantitative trait found to be associated with ASD [
28,
29]. A familial study showed that plasma free serotonin is correlated with ASD but is not different between ASD cases and their siblings or parents [
30]. This study also pointed out that the difference in serotonin is due only to the serotonin of the platelet fraction as the association was not found for serotonin levels in platelet-poor plasma. A study by Piven J et al. showed that serotonin levels in platelet-rich plasma of ASD patients with affected siblings were significantly higher than probands without affected siblings, though, the latter cases still had serotonin levels significantly higher than controls [
31]. A recent meta-analysis of all literature studies reporting ASD and control blood serotonin values concluded that elevated serotonin levels were recorded in 28.3 % in whole blood and 22.5 % in platelet-rich plasma samples of ASD patients, as reported in 15 and 4 studies, respectively, using different detection techniques [
32]. It is difficult to make an overall conclusion concerning the true association between ASD and serotonin as potential biomarker since serotonin concentrations are highly dynamic in the central nervous system and around [
33], and different methods to measure serotonin using different biomaterials (whole blood, platelets, platelet-rich, or platelet-poor plasma) do exist [
34]. This, altogether, probably accounts for the differences amongst a large number of studies that report serotonin levels as being a possible true biomarker for ASD or not.
Platelets store and release upon activation of serotonin from their dense granules and therefore, we hypothesized that differences in the platelet serotonin metabolism can be due to abnormalities in platelet dense granule formation, transport, and secretion. Therefore, it is also important to study the morphology and release of the platelet dense granules in addition to measuring serotonin levels. Indeed, we previously observed an altered platelet dense granule morphology in three unrelated ASD patients with chromosomal translocations. It was shown that the identified ASD candidate genes
NBEA,
SCAMP5, and
AMISYN involved in these translocations play a role in stimulated secretion of large dense-core vesicles [
35,
36]. Interestingly, heterozygous NBEA-deficient mice also have autistic-like behavior problems [
37] and a platelet dense granule morphology defect but these mice have normal serotonin levels in platelets and serum [
38]. Therefore, we hypothesized that in at least a subgroup of ASD patients, a defect in synaptic neuronal signaling would be reflected by alterations in platelet dense granule secretion. Platelets offer a unique opportunity to assess the stimulated dense granule function, in a relatively noninvasive and high-throughput manner because they can be isolated from peripheral blood in a non-active state. We here evaluated platelet count, size, and activation in addition to the classic measurements of only plasma serotonin levels in a non-syndromic familial ASD cohort.