Effects of low-frequency stimulation of the subthalamic nucleus on movement in Parkinson's disease

Excessive synchronization of basal ganglia neural activity at low frequencies is considered a hallmark of Parkinson's disease (PD). However, few studies have unambiguously linked this activity to movement impairment through direct stimulation of basal ganglia targets at low frequency. Furthermore, these studies have varied in their methodology and findings, so it remains unclear whether stimulation at any or all frequencies ≤ 20 Hz impairs movement and if so, whether effects are identical across this broad frequency band. To address these issues, 18 PD patients chronically implanted with deep brain stimulation (DBS) electrodes in both subthalamic nuclei were stimulated bilaterally at 5, 10 and 20 Hz after overnight withdrawal of their medication and the effects of the DBS on a finger tapping task were compared to performance without DBS (0 Hz). Tapping rate decreased at 5 and 20 Hz compared to 0 Hz (by 11.8 ± 4.9%, p = 0.022 and 7.4 ± 2.6%, p = 0.009, respectively) on those sides with relatively preserved baseline task performance. Moreover, the coefficient of variation of tap intervals increased at 5 and 10 Hz compared to 0 Hz (by 70.4 ± 35.8%, p = 0.038 and 81.5 ± 48.2%, p = 0.043, respectively). These data suggest that the susceptibility of basal ganglia networks to the effects of excessive synchronization may be elevated across a broad low-frequency band in parkinsonian patients, although the nature of the consequent motor impairment may depend on the precise frequencies at which synchronization occurs.
Introduction
There is extensive evidence that neuronal activity is abnormally synchronized at low frequencies in Parkinson's disease (PD) and in animal models of parkinsonism (reviewed in Gatev et al., 2006; Hammond et al., 2007; Uhlhaas and Singer, 2006). However, this does not, by itself, prove that pathological synchrony is mechanistically important in parkinsonism. More persuasive evidence would be the impairment of voluntary movement by the artificial synchronization of neural activity in the basal ganglia. Such synchronization is possible by stimulating deep brain electrodes implanted for the treatment of PD at low frequencies, rather than at those frequencies above 100 Hz used for therapeutic benefit. Electrical stimulation of surgical targets like the subthalamic nucleus (STN) simultaneously activates neural elements in the vicinity of the electrode and this synchronous activity is then propagated onwards, as evinced by evoked pallidal (Brown et al., 2004; Hashimoto et al., 2003), cortical (MacKinnon et al., 2005) and muscular activity (Ashby et al., 1999, 2001).
So far there have been several reports of the impairment of movement by stimulation of the STN at frequencies ≤ 20 Hz in patients with PD. Moro et al. (2002) and Chen et al. (2007) studied finger tapping during DBS at 5 Hz and 20 Hz, respectively, and found this to be slowed. However, Timmermann et al. (2004), using the motor United Parkinson's Disease rating scale (UPDRS), failed to confirm a worsening during DBS at 5 and 20 Hz, but did find increased bradykinesia with stimulation at 10 Hz. Another study evaluated tapping performance over several frequencies within the same patients, but only found weak effects that involved relative rather than absolute impairments in motor performance, superimposed upon an overall tendency for movement to improve with increasing stimulation frequency (Fogelson et al., 2005). Accordingly, it is unclear whether stimulation at any or all frequencies ≤ 20 Hz impairs movement and if so, whether effects are identical across this broad frequency band. The issue is an important one, as although spontaneous synchrony tends to occur at frequencies centered around 20 Hz in PD (Hammond et al., 2007), it occurs at rather lower frequencies in the 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) primate model of PD (Goldberg et al., 2004; Raz et al., 1996, 2000).
Here we contrast the effects of STN stimulation and thereby extrinsically imposed synchronization at a number of frequencies ≤ 20 Hz, to establish whether all such frequencies impair movement and, if so, whether they impair movement in the same way. To this end we studied performance in a simple finger tapping task, as this is objective and correlates with motor impairment (Giovannoni et al., 1999; Rabey et al., 2002), and considered changes in task execution according to baseline performance (Chen et al., 2006a).
Materials and methods
Patients and surgery
Twenty patients participated with informed consent and the permission of the local ethics committees (5 females, mean age 59.5 ± 1.4 years; mean disease duration 13.5 ± 1.0 years). Their clinical details are summarized in Table 1. Fourteen of these patients had also been recorded at least 6 months previously in a different paradigm involving stimulation at 20 Hz, 50 Hz and 130 Hz (Chen et al., 2007). Implantation of bilateral STN DBS electrodes was performed in all subjects for treatment of Parkinson's disease at least 6 months prior to study (mean 34.7 ± 5.9 months). The DBS electrode used was model 3389 (Medtronic Neurological Division, Minneapolis, USA) with four platinum–iridium cylindrical surfaces (1.27 mm diameter and 1.5 mm length) and a centre-to-centre separation of 2 mm. Contact 0 was the most caudal and contact 3 was the most rostral. The intended coordinates at the tip of contact 0 were 10–12 mm from the midline, 0–2 mm behind the midcommissural point and 3–5 mm below the anterior commissural–posterior commissural line. Adjustments to the intended coordinates were made in accordance with the direct visualization of STN in individual stereotactic MRI (Hariz et al., 2003) and, in the patients operated in Taiwan (n = 7), the results of microelectrode recordings. Correct placement of the DBS electrodes in the region of the STN was further supported by: [1] effective intra-operative macrostimulation; [2] post-operative T2-weighted MRI compatible with the placement of at least one electrode contact in the STN region; [3] significant improvement in UPDRS motor score during chronic DBS off medication (22.7 ± 3.0) compared to UPDRS off medication with stimulator switched off (52.6 ± 4.8; p < 0.00001, paired t-test). One patient was excluded due to the absence of significant improvement in UPDRS motor score during chronic DBS and another one due to missing clinical data.
Protocol
All patients were assessed after overnight withdrawal of antiparkinsonian medication, although the long action of many of the drugs used to treat PD meant that patients may still have been partially treated when assessed. They were studied when the stimulator was switched off and during bilateral STN stimulation at 5 Hz, 10 Hz and 20 Hz. The stimulation types were assessed in pseudo-randomized order across patients, as was the presentation order of trials within a stimulation type. Stimulation contacts, amplitude and pulse duration were the same as utilized for therapeutic high frequency stimulation in each subject (see Table 1). There was no evidence of capsular spread during stimulation, as determined by clinical examination. Patients were not informed of the stimulation type. We did not stimulate one side at a time to avoid possible functional compensation by the non-stimulated side. We waited 20 min after changing between conditions before testing. This is sufficient time to elicit about 75% of DBS effects (Temperli et al., 2003).
Task
The task was repetitive depression of a keyboard key as fast as possible by rapid alternating flexion and extension of the index finger at the level of the metacarpophalangeal joint (Chen et al., 2006a, 2007). Tapping was performed in two runs of 30 s, separated by ∼ 30-s rest and each hand tested separately (giving four runs per condition). Data from one side were rejected as these were collected contralateral to a previous unilateral pallidotomy (case 18 in Table 1). The number of taps made with the index finger in 30 s was recorded and the run from each pair with the best performance selected for analysis, as this was less likely to be affected by fatigue, or the effects of impaired arousal/concentration.
Statistics
The results of the tapping task in patients were analyzed according to their baseline performance (e.g. without stimulation). The lower limit of normal baseline performance was obtained by testing ten healthy age matched control subjects (20 sides, 4 males, mean age 57 years, range 52–64 years) using the same tapping task. The mean tapping rate in this control group was 162 taps/30 s. The lower limit of the normal range (e.g. mean − [2 × standard deviation]) in this control group was 127 taps per 30 s. The 35 tapping sides studied in the 18 patients were accordingly divided into those with baseline performance within normal limits (n = 17; the mean tapping performance across this group, 157 taps/30 s, was still lower than the mean tapping performance in healthy subjects) and those with baseline tapping rates lower than normal limits (n = 18; mean tapping performance 58 taps/30 s). The rationale behind this approach was to select those sides (with baseline performance within normal limits) in which any deleterious effects of DBS would not be overshadowed by the beneficial effects of DBS-induced suppression of spontaneous pathological activity or limited by floor effects due to major baseline impairment (Chen et al., 2006a, 2007). Four subjects had sides distributed across the two groups of differing baseline tapping performance. Tapping rates and coefficients of variation were normally distributed (one-sample Kolmogorov–Smirnov tests, p > 0.05). Repeated-measures ANOVAs with within-subjects simple contrasts (comparing different frequencies of stimulation to no stimulation) were performed in SPSS (SPSS for Windows version 11, SPSS Inc., Chicago, IL, USA). Mauchly's test was used to determine the sphericity of the data entered in the ANOVAs, and where data were non-spherical Greenhouse–Geisser corrections applied. Means ± standard error of the means are presented in the text.
Results
Low-frequency stimulation had no reliable clinical effect and did not consistently induce tremor, mobile dyskinesia, or dystonic posturing. Four patients experienced dystonic postures during the experiment and one had increased tremor. However, these effects were seen for overlapping stimulation frequencies. We divided the tapping sides into two groups according to whether or not tapping performance off DBS was within normal limits established on 20 sides in 10 healthy age-matched subjects (see Materials and methods). ANOVA of tapping scores with factors FREQUENCY (four levels: 0, 5, 10 and 20 Hz) and BASELINE TAPPING PERFORMANCE (two levels: within normal limits and less than normal limits) demonstrated a within-subjects interaction between FREQUENCY and BASELINE TAPPING PERFORMANCE (F[3,48] = 4.224, p = 0.01). Data were further analyzed with separate ANOVAs in each baseline tapping performance group. In those patients with baseline tapping performance within normal limits, ANOVA with the factor FREQUENCY confirmed that the latter was a significant main effect (F[3,48] = 3.777, p = 0.016). Within-subjects contrasts indicated that tapping during 5 and 20 Hz stimulation was worse than during no stimulation (F[1,16] = 6.385, p = 0.022 and F[1,16] =8.793, p = 0.009, respectively). The average deterioration in tapping rate during 5 and 20 Hz stimulation compared to no stimulation (0 Hz) in this group was 11.8 ± 4.9% and 7.4 ± 2.6%, respectively (Fig. 1). There was a trend towards a decreased tapping performance at 10 Hz compared to 0 Hz (F[1,16] = 3.578, p = 0.077). There was no effect of FREQUENCY in patients with baseline tapping performance below normal limits (ANOVA, F[1.9,32.8] = 2.202, p = 0.128).
We also analyzed the variability in tapping as measured by the coefficient of variation (CV) of the time intervals between successive taps on those sides with baseline tapping performance within normal limits. ANOVA with the factor FREQUENCY (four levels: 0, 5, 10 and 20 Hz) revealed a significant effect of FREQUENCY (F[3,48] = 3.408, p = 0.025). Within-subjects contrasts indicated that the CV increased during 5 and 10 Hz stimulation compared with no stimulation (F[1,16] = 5.144, p = 0.038 and F[1,16] = 4.852, p = 0.043, respectively). The average increase of the CV during 5 and 10 Hz stimulation compared to 0 Hz in this group was 70.4 ± 35.8% and 81.5 ± 48.2%, respectively (Fig. 2). There was no difference between the CV at 20 Hz compared to 0 Hz (F[1,16] = 0.871, p = 0.365). There was, however, a trend for the CV with 5 Hz stimulation to exceed that with 20 Hz stimulation (t-test; p = 0.059).
Discussion
We have shown that STN DBS at a variety of low frequencies can slow distal upper limb movements in PD patients with relatively preserved baseline tapping function at the time of study. The effect was present with DBS at 5 Hz and 20 Hz in line with previous studies (Chen et al., 2007; Fogelson et al., 2005; Moro et al., 2002), and there was a trend towards a similar effect with stimulation at 10 Hz (Timmermann et al., 2004). These effects were apparent when tapping sides were separately analyzed according to whether the level of baseline performance was within or outside of normal limits, in line with previous studies suggesting that deleterious effects of DBS are more evident on those sides with relatively preserved baseline performance (Chen et al., 2006a, 2007). The effect was not apparent during stimulation on those sides with impaired baseline performance, either because of confounding, albeit mild, suppressive effects of low-frequency DBS on spontaneous pathological oscillations or because of floor effects (Chen et al., 2007; Fogelson et al., 2005).
In principle, then, the susceptibility of basal ganglia–cortical loops to the effects of excessive synchronization may be elevated across a broad low-frequency band in parkinsonian patients. Accordingly, the relatively different frequency ranges of pathological synchronization in patients and MPTP-treated primates (Hammond et al., 2007) may be more indicative of the resonance properties of basal ganglia networks in the different situations, rather than any fundamental differences in the mechanism of bradykinesia. However, it must be stressed that this is a generalization, and although synchronization at different frequencies may conspire to disturb movement, there may still be subtle differences in the way movement is impaired. This is brought out by the differential effects of low-frequency stimulation on the variation in tapping intervals, evident in differences in the coefficient of variation and hence independent of any differences in tapping rate. Only DBS at 5 and 10 Hz increased temporal variability, whereas DBS at 20 Hz selectively decreased tapping rates without changing tapping variability.
The implication is that basal ganglia networks are involved in processing related to the temporal patterning and regularity of movement and that these circuits may be particularly susceptible to disruption by pathological synchronization at frequencies ≤ 10 Hz. In support of basal ganglia involvement in the temporal patterning of movement, PD patients have increased temporal variability in finger tapping (Giovannoni et al., 1999; Shimoyama et al., 1990), and temporal variability in motor performance is a very early feature of Huntington's disease (Hinton et al., 2007). Indeed, Flowers considered increased variability of movement in both time and space to be one of the core components of motor dysfunction in PD, along with a basic slowness of movement, and a difficulty in initiating and maintaining movement (Sheridan et al., 1987). This variability in motor performance may also relate to the phenomenon of freezing. No overt freezing episodes were observed during tapping in our patients, but an increased variability of stride has been shown in PD patients experiencing freezing of gait independent of frank freezing episodes (Hausdorff et al., 2003).
However, a primary disturbance of temporal patterning is not the only potential interpretation for the increased variability seen during stimulation at 5 Hz and 10 Hz. Tremor was not seen during low-frequency stimulation (except in one patient), in agreement with Timmermann et al. (2004), nor were there any obvious and consistent dyskinesias. Nevertheless, it is possible that synchronization at frequencies ≤ 10 Hz induced subtle hyperkinesias that led to increased temporal variability across taps. A previous case report describes dyskinetic movements induced by STN DBS at 5 Hz (Liu et al., 2002a), and there is increasing evidence that excessive synchronization over 4–10 Hz within basal ganglia circuits may be related to both levodopa-induced dyskinesias in PD (Alonso-Frech et al., 2006) and mobile elements of dystonia (Chen et al., 2006b; Liu et al., 2002b; Silberstein et al., 2003). Relevant in this regard, a recent study demonstrated an increased variability of speech rate in patients treated with l-DOPA and suggested that this effect was related to dyskinesia (De Letter et al., 2006). Variability of swing movement was also observed in the gait of dyskinetic CP children (Abel et al., 2003).
In summary, our results provide further evidence that DBS of the STN over a relatively broad band of low frequencies can impair movement, in line with other more circumstantial evidence of an association between low-frequency synchrony in basal ganglia–cortical loops and altered movement (see recent reviews by Gatev et al., 2006; Uhlhaas and Singer, 2006; Hammond et al., 2007). The present results also raise the important possibility that the detailed profile of motor abnormalities evident in extrapyramidal diseases depends to some extent on the precise frequencies at which pathological synchronization occurs. Indeed, some differences in the details of the effects of pathological synchrony at different frequencies might be anticipated, given the evidence for selective tuning of basal ganglia–cortical sub-circuits (Fogelson et al., 2006).

parkinson's disease
synchronization
basal ganglia
dbs


Degeneration of the intervertebral disc

The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry, but morphologically it is clearly different. It shows degenerative and ageing changes earlier than does any other connective tissue in the body. It is believed to be important clinically because there is an association of disc degeneration with back pain. Current treatments are predominantly conservative or, less commonly, surgical; in many cases there is no clear diagnosis and therapy is considered inadequate. New developments, such as genetic and biological approaches, may allow better diagnosis and treatments in the future.
Introduction
Back pain is a major public health problem in Western industrialized societies. It causes suffering and distress to patients and their families, and affects a large number of people; the point prevalence rates in a number of studies ranged from 12% to 35% [1], with around 10% of sufferers becoming chronically disabled. It also places an enormous economic burden on society; its total cost, including direct medical costs, insurance, lost production and disability benefits, is estimated at €12 billion per annum in the UK and 1.7% of the gross national product in The Netherlands [1,2].
Back pain is strongly associated with degeneration of the intervertebral disc [3]. Disc degeneration, although in many cases asymptomatic [4], is also associated with sciatica and disc herniation or prolapse. It alters disc height and the mechanics of the rest of the spinal column, possibly adversely affecting the behaviour of other spinal structures such as muscles and ligaments. In the long term it can lead to spinal stenosis, a major cause of pain and disability in the elderly; its incidence is rising exponentially with current demographic changes and an increased aged population.
Discs degenerate far earlier than do other musculoskeletal tissues; the first unequivocal findings of degeneration in the lumbar discs are seen in the age group 11–16 years [5]. About 20% of people in their teens have discs with mild signs of degeneration; degeneration increases steeply with age, particularly in males, so that around 10% of 50-year-old discs and 60% of 70-year-old discs are severely degenerate [6].
In this short review we outline the morphology and biochemistry of normal discs and the changes that arise during degeneration. We review recent advances in our understanding of the aetiology of this disorder and discuss new approaches to treatment.
Disc morphology
The normal disc
The intervertebral discs lie between the vertebral bodies, linking them together (Fig. 1). They are the main joints of the spinal column and occupy one-third of its height. Their major role is mechanical, as they constantly transmit loads arising from body weight and muscle activity through the spinal column. They provide flexibility to this, allowing bending, flexion and torsion. They are approximately 7–10 mm thick and 4 cm in diameter (anterior–posterior plane) in the lumbar region of the spine [7,8]. The intervertebral discs are complex structures that consist of a thick outer ring of fibrous cartilage termed the annulus fibrosus, which surrounds a more gelatinous core known as the nucleus pulposus; the nucleus pulposus is sandwiched inferiorly and superiorly by cartilage end-plates.
The central nucleus pulposus contains collagen fibres, which are organised randomly [9], and elastin fibres (sometimes up to 150 μm in length), which are arranged radially [10]; these fibres are embedded in a highly hydrated aggrecan-containing gel. Interspersed at a low density (approximately 5000/mm3 [11]) are chondrocyte-like cells, sometimes sitting in a capsule within the matrix. Outside the nucleus is the annulus fibrosus, with the boundary between the two regions being very distinct in the young individual (<10 years).
The annulus is made up of a series of 15–25 concentric rings, or lamellae [12], with the collagen fibres lying parallel within each lamella. The fibres are orientated at approximately 60° to the vertical axis, alternating to the left and right of it in adjacent lamellae. Elastin fibres lie between the lamellae, possibly helping the disc to return to its original arrangement following bending, whether it be flexion or extension. They may also bind the lamellae together as elastin fibres pass radially from one lamella to the next [10]. The cells of the annulus, particularly in the outer region, tend to be fibroblast-like, elongated, thin and aligned parallel to the collagen fibres. Toward the inner annulus the cells can be more oval. Cells of the disc, both in the annulus and nucleus, can have several long, thin cytoplasmic projections, which may be more than 30 μm long [13,14] (WEB Johnson, personal communication). Such features are not seen in cells of articular cartilage [13]. Their function in disc is unknown but it has been suggested that they may act as sensors and communicators of mechanical strain within the tissue [13].
The third morphologically distinct region is the cartilage end-plate, a thin horizontal layer, usually less than 1 mm thick, of hyaline cartilage. This interfaces the disc and the vertebral body. The collagen fibres within it run horizontal and parallel to the vertebral bodies, with the fibres continuing into the disc [8].
The healthy adult disc has few (if any) blood vessels, but it has some nerves, mainly restricted to the outer lamellae, some of which terminate in proprioceptors [15]. The cartilaginous end-plate, like other hyaline cartilages, is normally totally avascular and aneural in the healthy adult. Blood vessels present in the longitudinal ligaments adjacent to the disc and in young cartilage end-plates (less than about 12 months old) are branches of the spinal artery [16]. Nerves in the disc have been demonstrated, often accompanying these vessels, but they can also occur independently, being branches of the sinuvertebral nerve or derived from the ventral rami or grey rami communicantes. Some of the nerves in discs also have glial support cells, or Schwann cells, alongside them [17].
Degenerated discs
During growth and skeletal maturation the boundary between annulus and nucleus becomes less obvious, and with increasing age the nucleus generally becomes more fibrotic and less gel-like [18]. With increasing age and degeneration the disc changes in morphology, becoming more and more disorganized (Fig. 2). Often the annular lamellae become irregular, bifurcating and interdigitating, and the collagen and elastin networks also appear to become more disorganised (J Yu, personal communication).
There is frequently cleft formation with fissures forming within the disc, particularly in the nucleus. Nerves and blood vessels are increasingly found with degeneration [15]. Cell proliferation occurs, leading to cluster formation, particularly in the nucleus [19,20]. Cell death also occurs, with the presence of cells with necrotic and apoptotic appearance [21,22]. These mechanisms are apparently very common; it has been reported that more than 50% of cells in adult discs are necrotic [21]. The morphological changes associated with disc degeneration were comprehensively reviewed recently by Boos et al. [5], who demonstrated an age-associated change in morphology, with discs from individuals as young as 2 years of age having some very mild cleft formation and granular changes to the nucleus. With increasing age comes an increased incidence of degenerative changes, including cell death, cell proliferation, mucous degeneration, granular change and concentric tears. It is difficult to differentiate changes that occur solely due to ageing from those that might be considered 'pathological'.
Biochemistry
Normal discs
The mechanical functions of the disc are served by the extracellular matrix; its composition and organization govern the disc's mechanical responses. The main mechanical role is provided by the two major macromolecular components. The collagen network, formed mostly of type I and type II collagen fibrils and making up approximately 70% and 20% of the dry weight of the annulus and nucleus, respectively [23], provides tensile strength to the disc and anchors the tissue to the bone. Aggrecan, the major proteoglycan of the disc [24], is responsible for maintaining tissue hydration through the osmotic pressure provided by its constituent chondroitin and keratan sulphate chains [25]. The proteoglycan and water content of the nucleus (around 50% and 80% of the wet weight, respectively) is greater than in the annulus (approximately 20% and 70% of the wet weight, respectively). In addition, there are many other minor components, such as collagen types III, V, VI, IX, X, XI, XII and XIV; small proteoglycans such as lumican, biglycan, decorin and fibromodulin; and other glycoproteins such as fibronectin and amyloid [26,27]. The functional role of many of these additional matrix proteins and glycoproteins is not yet clear. Collagen IX, however, is thought to be involved in forming cross-links between collagen fibrils and is thus important in maintaining network integrity [28].
The matrix is a dynamic structure. Its molecules are continually being broken down by proteinases such as the matrix metalloproteinases (MMPs) and aggrecanases, which are also synthesized by disc cells [29-31]. The balance between synthesis, breakdown and accumulation of matrix macromolecules determines the quality and integrity of the matrix, and thus the mechanical behaviour of the disc itself. The integrity of the matrix is also important for maintaining the relatively avascular and aneural nature of the healthy disc.
The intervertebral disc is often likened to articular cartilage, and indeed it does resemble it in many ways, particularly in the biochemical components present. However, there are significant differences between the two tissues, one of these being the composition and structure of aggrecan. Disc aggrecan is more highly substituted with keratan sulphate than that found in the deep zone of articular cartilage. In addition, the aggrecan molecules are less aggregated (30%) and more heterogeneous, with smaller, more degraded fragments in the disc than in articular cartilage (80% aggregated) from the same individual [32]. Disc proteoglycans become increasingly difficult to extract from the matrix with increasing age [24]; this may be due to extensive cross-linking, which appears to occur more within the disc matrix than in other connective tissues.
Changes in disc biochemistry with degeneration
The most significant biochemical change to occur in disc degeneration is loss of proteoglycan [33]. The aggrecan molecules become degraded, with smaller fragments being able to leach from the tissue more readily than larger portions. This results in loss of glycosaminoglycans; this loss is responsible for a fall in the osmotic pressure of the disc matrix and so a loss of hydration.
Even in degenerate discs, however, the disc cells can retain the ability to synthesize large aggrecan molecules, with intact hyaluronan-binding regions, which have the potential to form aggregates [24]. Less is known of how the small proteoglycan population changes with disc degeneration, although there is some evidence that the amount of decorin, and more particularly biglycan, is elevated in degenerate human discs as compared with normal ones [34].
Although the collagen population of the disc also changes with degeneration of the matrix, the changes are not as obvious as those of the proteoglycans. The absolute quantity of collagen changes little but the types and distribution of collagens can alter. For example, there may be a shift in proportions of types of collagens found and in their apparent distribution within the matrix. In addition, the fibrillar collagens, such as type II collagen, become more denatured, apparently because of enzymic activity. As with proteoglycans, the triple helices of the collagens are more denatured and ruptured than are those found in articular cartilage from the same individual; the amount of denatured type II collagen increases with degeneration [35,36]. However, collagen cross-link studies indicate that, as with proteoglycans, new collagen molecules may be synthesized, at least early in disc degeneration, possibly in an attempt at repair [37].
Other components can change in disc degeneration and disease in either quantity or distribution. For example, fibronectin content increases with increasing degeneration and it becomes more fragmented [38]. These elevated levels of fibronectin could reflect the response of the cell to an altered environment. Whatever the cause, the formation of fibronectin fragments can then feed into the degenerative cascade because they have been shown to downregulate aggrecan synthesis but to upregulate the production of some MMPs in in vitro systems.
The biochemistry of disc degeneration indicates that enzymatic activity contributes to this disorder, with increased fragmentation of the collagen, proteoglycan and fibronectin populations. Several families of enzymes are capable of breaking down the various matrix molecules of disc, including cathepsins, MMPs and aggrecanases. Cathepsins have maximal activity in acid conditions (e.g. cathepsin D is inactive above pH 7.2). In contrast, MMPs and aggrecanases have an optimal pH that is approximately neutral. All of these enzymes have been identified in disc, with higher levels of, for example, MMPs in more degenerate discs [39]. Cathepsins D and L and several types of MMPs (MMP-1, -2, -3, -7, -8, -9 and -13) occur in human discs; they may be produced by the cells of the disc themselves as well as by the cells of the invading blood vessels. Aggrecanases have also been shown to occur in human disc but their activity is apparently less obvious, at least in more advanced disc degeneration [29,30,40].
Effect of degenerative changes on disc function and pathology
The loss of proteoglycan in degenerate discs [33] has a major effect on the disc's load-bearing behaviour. With loss of proteoglycan, the osmotic pressure of the disc falls [41] and the disc is less able to maintain hydration under load; degenerate discs have a lower water content than do normal age-matched discs [33], and when loaded they lose height [42] and fluid more rapidly, and the discs tend to bulge. Loss of proteoglycan and matrix disorganization have other important mechanical effects; because of the subsequent loss of hydration, degenerated discs no longer behave hydrostatically under load [43]. Loading may thus lead to inappropriate stress concentrations along the end-plate or in the annulus; the stress concentrations seen in degenerate discs have also been associated with discogenic pain produced during discography [44].
Such major changes in disc behaviour have a strong influence on other spinal structures, and may affect their function and predispose them to injury. For instance, as a result of the rapid loss of disc height under load in degenerate discs, apophyseal joints adjacent to such discs (Fig. 1) may be subject to abnormal loads [45] and eventually develop osteoarthritic changes. Loss of disc height can also affect other structures. It reduces the tensional forces on the ligamentum flavum and hence may cause remodelling and thickening. With consequent loss of elasticity [46], the ligament will tend to bulge into the spinal canal, leading to spinal stenosis – an increasing problem as the population ages.
Loss of proteoglycans also influences the movement of molecules into and out of the disc. Aggrecan, because of its high concentration and charge in the normal disc, prevents movement of large uncharged molecules such as serum proteins and cytokines into and through the matrix [47]. The fall in concentration of aggrecan in degeneration could thus facilitate loss of small, but osmotically active, aggrecan fragments from the disc, possibly accelerating a degenerative cascade. In addition, loss of aggrecan would allow increased penetration of large molecules such as growth factor complexes and cytokines into the disc, affecting cellular behaviour and possibly the progression of degeneration. The increased vascular and neural ingrowth seen in degenerate discs and associated with chronic back pain [48] is also probably associated with proteoglycan loss because disc aggrecan has been shown to inhibit neural ingrowth [49,50].
Disc herniation
The most common disc disorder presenting to spinal surgeons is herniated or prolapsed intervertebral disc. In these cases the discs bulge or rupture (either partially or totally) posteriorly or posterolaterally, and press on the nerve roots in the spinal canal (Fig. 1). Although herniation is often thought to be the result of a mechanically induced rupture, it can only be induced in vitro in healthy discs by mechanical forces larger than those that are ever normally encountered; in most experimental tests, the vertebral body fails rather than the disc [51]. Some degenerative changes seem necessary before the disc can herniate; indeed, examination of autopsy or surgical specimens suggest that sequestration or herniation results from the migration of isolated, degenerate fragments of nucleus pulposus through pre-existing tears in the annulus fibrosus [52].
It is now clear that herniation-induced pressure on the nerve root cannot alone be the cause of pain because more than 70% of 'normal', asymptomatic people have disc prolapses pressurizing the nerve roots but no pain [4,53]. A past and current hypothesis is that, in symptomatic individuals, the nerves are somehow sensitized to the pressure [54], possibly by molecules arising from an inflammatory cascade from arachodonic acid through to prostaglandin E2, thromboxane, phospholipase A2, tumour necrosis factor-α, the interleukins and MMPs. These molecules can be produced by cells of herniated discs [55], and because of the close physical contact between the nerve root and disc following herniation they may be able to sensitize the nerve root [56,57]. The exact sequence of events and specific molecules that are involved have not been identified, but a pilot study of sciatic patients treated with tumour necrosis factor-α antagonists is encouraging and supports this proposed mechanism [58,59]. However, care must be exercised in interrupting the inflammatory cascade, which can also have beneficial effects. Molecules such as MMPs, which are produced extensively in prolapsed discs [30], almost certainly play a major role in the natural history of resorbing the offending herniation.
Aetiology of disc degeneration
Disc degeneration has proved a difficult entity to study; its definition is vague, with diffuse parameters that are not always easy to quantify. In addition, there is a lack of a good animal model. There are significant anatomical differences between humans and the laboratory animals that are traditionally used as models of other disorders. In particular, the nucleus differs; in rodents as well as many other mammals, the nucleus is populated by notochordal cells throughout adulthood, whereas these cells disappear from the human nucleus after infancy [60]. In addition, although the cartilage end-plate in humans acts as a growth plate for the vertebral body, in most animals the vertebrae have two growth plates within the vertebral body itself, and the cartilage end-plate is a much thinner layer than that found in humans. Thus, although the study of animals that develop degeneration spontaneously [61,62] and of injury models of degeneration [63,64] have provided some insight into the degenerative processes, most information on aetiology of disc degeneration to date has come from human studies.
Nutritional pathways to disc degeneration
One of the primary causes of disc degeneration is thought to be failure of the nutrient supply to the disc cells [65]. Like all cell types, the cells of the disc require nutrients such as glucose and oxygen to remain alive and active. In vitro, the activity of disc cells is very sensitive to extracellular oxygen and pH, with matrix synthesis rates falling steeply at acidic pH and at low oxygen concentrations [66,67], and the cells do not survive prolonged exposure to low pH or glucose concentrations [68]. A fall in nutrient supply that leads to a lowering of oxygen tension or of pH (arising from raised lactic acid concentrations) could thus affect the ability of disc cells to synthesize and maintain the disc's extracellular matrix and could ultimately lead to disc degeneration.
The disc is large and avascular and the cells depend on blood vessels at their margins to supply nutrients and remove metabolic waste [69]. The pathway from the blood supply to the nucleus cells is precarious because these cells are supplied virtually entirely by capillaries that originate in the vertebral bodies, penetrating the subchondral plate and terminating just above the cartilaginous end-plate [16,70]. Nutrients must then diffuse from the capillaries through the cartilaginous end-plate and the dense extracellular matrix of the nucleus to the cells, which may be as far as 8 mm from the capillary bed.
The nutrient supply to the nucleus cells can be disturbed at several points. Factors that affect the blood supply to the vertebral body such as atherosclerosis [71,72], sickle cell anaemia, Caisson disease and Gaucher's disease [73] all appear to lead to a significant increase in disc degeneration. Long-term exercise or lack of it appears to have an effect on movement of nutrients into the disc, and thus on their concentration in the tissue [74,75]. The mechanism is not known but it has been suggested that exercise affects the architecture of the capillary bed at the disc–bone interface. Finally, even if the blood supply remains undisturbed, nutrients may not reach the disc cells if the cartilaginous end-plate calcifies [65,76]; intense calcification of the end-plate is seen in scoliotic discs [77], for instance. Disturbances in nutrient supply have been shown to affect transport of oxygen and lactic acid into and out of the disc experimentally [78] and in patients [79].
Although little information is available to relate nutrient supply to disc properties in patients, a relationship has been found between loss of cell viability and a fall in nutrient transport in scoliotic discs [80,81]. There is also some evidence that nutrient transport is affected in disc degeneration in vivo [82], and the transport of solutes from bone to disc measured in vitro was significantly lower in degenerate than in normal discs [65]. Thus, although there is as yet little direct evidence, it now seems apparent that a fall in nutrient supply will ultimately lead to degeneration of the disc.
Mechanical load and injury
Abnormal mechanical loads are also thought to provide a pathway to disc degeneration. For many decades it was suggested that a major cause of back problems is injury, often work-related, which causes structural damage. It is believed that such an injury initiates a pathway that leads to disc degeneration and finally to clinical symptoms and back pain [83]. Animal models have supported this finding. Although intense exercise does not appear to affect discs adversely [84] and discs are reported to respond to some long-term loading regimens by increasing proteoglycan content [85], experimental overloading [86] or injury to the disc [63,87] can induce degenerative changes. Further support for the role of abnormal mechanical forces in disc degeneration comes from findings that disc levels adjacent to a fused segment degenerate rapidly (for review [88]).
This injury model is also supported by many epidemiological studies that have found associations between environmental factors and development of disc degeneration and herniation, with heavy physical work, lifting, truck-driving, obesity and smoking found to be the major risk factors for back pain and degeneration [89-91]. As a result of these studies, there have been many ergonomic interventions in the workplace [91]. However, the incidence of disc degeneration-related disorders has continued to rise despite these interventions. Over the past decade, as magnetic resonance imaging has refined classifications of disc degeneration [5,92], it has become evident that, although factors such as occupation, psychosocial factors, benefit payments and environment are linked to disabling back pain [93,94], contrary to previous assumptions these factors have little influence on the pattern of disc degeneration itself [95,96]. This illustrates the tenuous relationship between degeneration and clinical symptoms.
Genetic factors in disc degeneration
More recent work suggested that the factors that lead to disc degeneration may have important genetic components. Several studies have reported a strong familial predisposition for disc degeneration and herniation [97-99]. Findings from two different twin studies conducted during the past decade showed heritability exceeding 60% [100,101]. Magnetic resonance images in identical twins, who were discordant for major risk factors such as smoking or heavy work, were very similar with respect to the spinal columns and the patterns of disc degeneration (Fig. 3) [102].
Genetic predisposition has been confirmed by recent findings of associations between disc degeneration and gene polymorphisms of matrix macromolecules. The approach to date has been via searching for candidate genes, with the main focus being extracellular matrix genes. Although there is a lack of association between disc degeneration and polymorphisms of the major collagens in the disc, collagen types I and II [103], mutations of two collagen type IX genes, namely COL9A2 and COL9A3, have been found to be strongly associated with lumbar disc degeneration and sciatica in a Finnish population [104,105]. The COL9A2 polymorphism is found only in a small percentage of the Finnish population, but all individuals with this allele had disc degenerative disorders, suggesting that it is associated with a dominantly inherited disease. In both these mutations, tryptophan (the most hydrophobic amino acid, which is not normally found in any collagenous domain) substituted for other amino acids, potentially affecting matrix properties [103].
Other genes associated with disc generation have also been identified. Individuals with a polymorphism in the aggrecan gene were found to be at risk for early disc degeneration in a Japanese study [106]. This mutation leads to aggrecan core proteins of different lengths, with an over-representation of core proteins able to bind only a low number of chondroitin sulfate chains among those with severe disc degeneration. Presumably these individuals have a lower chondroitin sulfate content than normal, and their discs will behave similarly to degenerate discs that have lost proteoglycan by other mechanisms. Studies of transgenic mice have also demonstrated that mutations in structural matrix molecules such as aggrecan [107], collagen II [108] and collagen IX [109] can lead to disc degeneration. Mutations in genes other than those of structural matrix macromolecules have also been associated with disc degeneration. A polymorphism in the promoter region of the MMP-3 gene was associated with rapid degeneration in elderly Japanese subjects [110]. In addition, two polymorphisms of the vitamin D receptor gene were the first mutations shown to be associated with disc degeneration [111-114]. The mechanism of vitamin D receptor gene polymorphism involvement in disc degeneration is unknown, but at present it does not appear to be related to differences in bone density [111,112,114].
All of the genetic mutations associated with disc degeneration to date have been found using a candidate gene approach and all, apart from the vitamin D receptor polymorphism, are concerned with molecules that determine the integrity and function of the extracellular matrix. However, mutations in other systems such as signalling or metabolic pathways could lead to changes in cellular activity that may ultimately result in disc degeneration [115]. Different approaches may be necessary to identify such polymorphisms. Genetic mapping, for instance, has identified a susceptibility locus for disc herniation, but the gene involved has not yet been identified [116].
In summary, the findings from these genetic and epidemiological studies point to the multifactorial nature of disc degeneration. It is evident now that mutations in several different classes of genes may cause the changes in matrix morphology, disc biochemistry and disc function typifying disc degeneration. Identification of the genes involved may lead to improved diagnostic criteria; for example, it is already apparent that the presence of specific polymorphisms increase the risk for disc bulge, annular tears, or osteophytes [112,117]. However, because of the evidence for gene–environment interactions [97,114,118], genetic studies in isolation are unlikely to delineate the various pathways of disc degeneration.
New therapies
Current treatments attempt to reduce pain rather than repair the degenerated disc. The treatments used presently are mainly conservative and palliative, and are aimed at returning patients to work. They range from bedrest (no longer recommended) to analgesia, the use of muscle relaxants or injection of corticosteroids, or local anaesthetic and manipulation therapies. Various interventions (e.g. intradiscal electrotherapy) are also used, but despite anecdotal statements of success trials thus far have found their use to be of little direct benefit [119]. Disc degeneration-related pain is also treated surgically either by discectomy or by immobilization of the affected vertebrae, but surgery is offered only to one in every 2000 back pain episodes in the UK; the incidence of surgical treatment is five times higher in the USA [93]. The success rates of all these procedures are generally similar. Although a recent study indicated that surgery improves the rate of recovery in well selected patients [120], 70–80% of patients with obvious surgical indications for back pain or disc herniation eventually recover, whether surgery is carried out or not [121,122].
Because disc degeneration is thought to lead to degeneration of adjacent tissues and be a risk factor in the development of spinal stenosis in the long term, new treatments are in development that are aimed at restoring disc height and biomechanical function. Some of the proposed biological therapies are outlined below.
Cell based therapies
The aim of these therapies is to achieve cellular repair of the degenerated disc matrix. One approach has been to stimulate the disc cells to produce more matrix. Growth factors can increase rates of matrix synthesis by up to fivefold [123,124]. In contrast, cytokines lead to matrix loss because they inhibit matrix synthesis while stimulating production of agents that are involved in tissue breakdown [125]. These proteins have thus provided targets for genetic engineering. Direct injection of growth factors or cytokine inhibitors has proved unsuccessful because their effectiveness in the disc is short-lived. Hence gene-therapy is now under investigation; it has the potential to maintain high levels of the relevant growth factor or inhibitor in the tissue. In gene therapy, the gene of interest (e.g. one responsible for producing a growth factor such as transforming growth factor-β or inhibiting interleukin-1) is introduced into target cells, which then continue to produce the relevant protein (for review [126]). This approach has been shown to be technically feasible in the disc, with gene transfer increasing transforming growth factor-β production by disc cells in a rabbit nearly sixfold [127]. However, this therapy is still far from clinical use. Apart from the technical problems of delivery of the genes into human disc cells, the correct choice of therapeutic genes requires an improved understanding of the pathogenesis of degeneration. In addition, the cell density in normal human discs is low, and many of the cells in degenerate discs are dead [21]; stimulation of the remaining cells may be insufficient to repair the matrix.
Cell implantation alone or in conjunction with gene therapy is an approach that may overcome the paucity of cells in a degenerate disc. Here, the cells of the degenerate disc are supplemented by adding new cells either on their own or together with an appropriate scaffold. This technique has been used successfully for articular cartilage [128,129] and has been attempted with some success in animal discs [130]. However, at present, no obvious source of clinically useful cells exists for the human disc, particularly for the nucleus, the region of most interest [131]. Moreover, conditions in degenerate discs, particularly if the nutritional pathway has been compromised [65], may not be favourable for survival of implanted cells. Nevertheless, autologous disc cell transfer has been used clinically in small groups of patients [132], with initial results reported to be promising, although few details of the patients or outcome measures are available.
At present, although experimental work demonstrates the potential of these cell-based therapies, several barriers prevent the use of these treatments clinically. Moreover, these treatments are unlikely to be appropriate for all patients; some method of selecting appropriate patients will be required if success with these therapies is to be realized.
Conclusion
Disorders associated with degeneration of the intervertebral disc impose an economic burden similar to that of coronary heart disease and greater than that of other major health problems such as diabetes, Alzheimer's disease and kidney diseases [1,133]. New imaging technologies, and advances in cell biology and genetics promise improved understanding of the aetiology, more specific diagnoses and targeted treatments for these costly and disabling conditions. However, the intervertebral disc is poorly researched, even in comparison with other musculoskeletal systems (Table 1). Moreover, the research effort in, for instance, the kidney in comparison with that in the disc is completely disparate to the relative costs of the disorders associated with each organ and the number of people affected. Unless more research attention is attracted to interverterbal disc biology, little will come from these new technologies, and back pain will remain as it is at present – a poorly diagnosed and poorly treated syndrome that reduces the quality of life of a significant proportion of the population.
Competing interests
None declared.
Abbreviations
MMP = matrix metalloproteinase.


What’s new in using platelet research? To unravel thrombopathies and other human disorders

This review on platelet research focuses on defects of adhesion, cytoskeletal organisation, signal transduction and secretion. Platelet defects can be studied by different laboratory platelet functional assays and morphological studies. Easy bruising or a suspected platelet-based bleeding disorder is of course the most obvious reason to test the platelet function in a patient. However, nowadays platelet research also contributes to our understanding of human pathology in other disciplines such as neurology, nephrology, endocrinology and metabolic diseases. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet research and disorders with a broader clinical phenotype. Classical thrombopathies involve disorders of platelet adhesion such as Glanzmann thrombastenia and Bernard-Soulier syndrome, defective G protein signalling diseases with impaired phospholipase C activation, and abnormal platelet granule secretion disorders such as gray platelet disorder and delta-storage pool disease. Other clinical symptoms besides a bleeding tendency have been described in MYH9-related disorders and Duchenne muscular dystrophy due to adhesion defects, and also in disorders of impaired Gs signalling, in Hermansky Pudlack disease and Chediak Higashi disease with abnormal secretion. Finally, platelet research can also be used to unravel novel mechanisms involved in many neurological disorders such as depression and autism with only a subclinical platelet defect.
Introduction
Normal hemostasis prevents spontaneous bleeding and traumatic hemorrhage by a coordinated sequence of cellular and biochemical reactions to the ultimate formation of a stable platelet-fibrin aggregate [20]. Platelets, under normal circumstances, circulate in close contact to the endothelial cell lining of the vessel wall, and respond to vascular damage by adhering to subendothelial structures. Platelet adhesion is the first step in the hemostatic plug formation [11]. The major platelet receptors involved in this process are the von Willebrand factor (vWF) receptor GP(glycoprotein)Ib/IX/V, the collagen integrin receptor α2β1, and the fibrinogen integrin receptor αIIBβ3 (Fig. 1a). Subsequent platelet spreading is conducted by cytoskeleton proteins including the structural subunit of the microtubules, the αβ-tubulin heterodimer, filamin and actin (Fig. 1a). The cytoskeleton is responsible for the shape of the resting platelet and carries out contractile events such as the secretion of granules and retraction of clots by activated cells.
Fig. 1a Schematic model of the main components involved in platelet adhesion and the cytoskeleton proteins. Platelet adhesion and its subsequent activation by calcium release is mainly regulated by the platelet receptor αIIbβ3 after binding to fibrinogen or the RGD domain of vWF, the main vWF receptor GPIb/IX/V and the collagen receptor α2β1. Microtubules together with the cytoplasmic, actin-rich cytoskeleton are responsible for the platelet structure. Different actin binding proteins have been identified in platelets such as filamin A, myosin and dystrophin. b Schematic model of G protein signal transduction in platelets regulated by Gq for platelet activation by the ultimate step of calcium release. Gi and Gs further influence the platelet activation by respectively inhibiting and stimulating the intracellular cAMP formation. c Schematic model of platelet secretion. The second amplification step in platelet activation is the release of alpha and dense granules in platelets guarantying irreversible platelet activation
Platelet adhesion also initiates multiple intracellular G protein-coupled signalling pathways (Fig. 1b). Stimulation of Gq by different ligands such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2) results in platelet activation by stimulating phospholipase C and a release of calcium from the intracellular stores. This platelet activation process is enhanced when Gi is activated and inhibited when Gs is activated both by modulating the intracellular cAMP level.
Platelet adhesion and activation eventually results in secretion from platelet organelles (Fig. 1c) [37]. Resting platelets circulate as discoid anuclear cells and consist of a lipid bilayer and an internal dense tubular system, where calcium is sequestered. The platelet cytoplasm contains mitochondria, glycogen particles, lysosomes, and the platelet-specific storage granules: the α-granules and dense granules (Fig. 2). The α-granules contain proteins such as platelet factor 4, β-thromboglobulin, platelet derived growth factor, fibrinogen, fibronectin, thrombospondin, plasminogen activator inhibitor I and vWF. Dense bodies are rich in serotonin, ATP, ADP and calcium.
Fig. 2Electron microscopy (original magnification ×22,500) of platelets showing the dense tubular system (DTS), microtubules (MT), open canalicular system (OCS), alpha granules (G), glycogen (Gly) and the dense bodies (DB)
The study of platelet adhesion, G protein signalling and secretion is particularly useful for our understanding of several clinical disorders (Table 1). The most obvious reason is of course the study of platelet defects in patients with an isolated platelet disorder leading to bleeding or thrombosis in order to gain additional information on the different pathways involved in platelet function. The analysis of human disorders in which the defective platelet phenotype is just one part of its clinical spectrum to unravel novel biological and genetic mechanisms involved in the disease. Also, using platelet functional and morphological studies can be used as a tool to find novel pathways involved in more complex disorders usually caused by more than one gene defect. Recently, it became obvious that the molecular pathways involved in more complex human disorders such as diabetes type 2 or neurological disorders such as schizophrenia, migraine, bipolar disorder, and depression can also be better understood by studying platelet signalling and secretion. Due to space limitations, it is impossible to give a full overview of all human disorders studied today by means of platelet research. This review describes some well-known as well as some less common disorders to illustrate how platelet research contributes to the understanding of thrombopathies [13, 35] as well as the broader future of this research outside its classical field of thrombosis and hemostasis (Table 1).
Table 1Syndromic and non-syndromic platelet defects and the implicated genes according to the defective platelet pathwayType of platelet defectIsolated platelet disordersDisorders including a platelet defectDisorders studied by functional platelet assaysAdhesion and cytoskeletal defectsGlanzmann thrombasteniaMay-Hegglin anomaly, Fechtner syndrome, Epstein syndrome, and Sebastian syndromeNeurological disorders as bipolar disorder, schizophrenia, depression, autismProlonged bleeding timeAll: macrothrombocytopenia, prolonged bleeding timeNo other clinical problemsAll: leucocyte inclusions, Epstein,Fechtner: nephritis, deafness, cataractsITGB3 or ITGA2MYH9Bernard-Soulier SyndromeDuchenne Muscular DystrophyMacrothrombocytopenia, prolonged bleeding timeProlonged bleeding after surgeryNo other clinical problemsMuscle degenerationGPIbα, GPIbβ or GPIXDMDG protein signalling defectsADP P2Y12 receptorInducible Gsα hyperfunction syndromeSubclinical platelet defectProlonged bleeding timeProlonged bleeding time after traumaNo other clinical problemsBrachydactyly, increased alkaline phosphatase and neurological or growth retardationP2Y12XLαsThromboxane TXA2 receptorPACAP overexpressionProlonged bleeding timeProlonged bleeding timeNo other clinical problemsMental retardation and hypogonadismTXA2RPACAPSecretion defectsGray platelet disorderHermansky Pudlack diseaseNeurological defect?Prolonged bleeding timeProlonged bleeding timeNo other clinical problemsAlbinism, lysosomal defect?HSP 1–8Delta storage Pool diseaseChediak Higashi diseaseProlonged bleeding timeProlonged bleeding timeNo other clinical problemsAlbinism, immunological lethal defect?LYSTThe following implicated genes are indicated in : ITGB3 integrin beta3; ITGA2 integrin alpha2; GPIbα glycoprotein Ibalpha; GPIbß glycoprotein Ibbeta; GPIX glycoprotein IX; P2Y12 purinergic receptor 12; TXASR thromboxane A2 receptor; MYH9 nonmuscle myosin heavy chain 9; DMD Dystrophin; XLαs extra-large stimulatory G protein alpha subunit; PACAP pituitary adenylate cylase-activating peptide; HSP 1–8 Hermansky Pudlack genes 1 through 8; LYST lysosomal trafficking regulator
When considering type of platelet tests
A clinical platelet-based bleeding problem is of course the main reason to investigate platelet function and morphology. The diagnostic approach to easy bruising or a suspected platelet-based bleeding disorder includes a careful history and physical examination of the patient as well as different laboratory investigations such as the Ivy bleeding time, platelet aggregation tests, ATP secretion, platelet adhesion by the platelet function analyzer (PFA100) and platelet morphology by electron microscopy [13, 40]. Reviewing the medical history can already establish whether the disorder is hereditary or acquired. The specific clinical findings useful in the differential diagnosis of coagulation versus platelet-based disorders are summarized in Table 2. Frequently, mucocutaneous bleedings characterize abnormalities of platelet function. In contrast, hemorrhage into synovial joints and deep muscular hemorrhage are signs of severe hereditary coagulation disorders and very rare events in disorders of platelets, vessels or acquired coagulation disorders. Inherited disorders of platelet function are further subdivided based on the functions or responses that are abnormal and therefore can belong to different subgroups including abnormal platelet adhesion, signalling and secretion [36]. Platelet-based bleeding disorders are usually classified according to abnormalities of platelet function, platelet number (thrombocytopenia) or both [36].
Table 2Clinical presentation of coagulation and platelet-based bleeding disordersClinical symptomsDisorders of coagulationDisorders of plateletsPetechiae, epistaxisRareCharacteristicSuperficial ecchymosesCommon: large and solitaryCharacteristic: small and multipleBleeding from superficial cuts and bruisesMinimalPersistant: often profuseDelayed bleedingCommonRareDeep dissecting hematomasCharacteristicRareHemarthrosisCharacteristicRare
Functional and morphological platelet studies in patients with mainly a neurological, metabolic or another clinical problem but no obvious bleeding problem are usually not performed for diagnostic purposes but rather for research aims. In such patients, novel insights are expected to result from the platelet research studies, which are still preliminary today but in the future hopefully will help to better define when to ask for what type of platelet tests in a given patient.
Thrombopathies
Glanzmann thrombasthenia and the Bernard-Soulier syndrome (BSS) are two rare inherited disorders of platelet adhesion. Glanzmann thrombasthenia (MIM 273800) is an autosomal recessive disorder, characterized by prolonged bleeding time and abnormal clot retraction [30, 31, 35]. The hallmark of this disease is severely reduced or absent platelet aggregation in response to various physiological platelet agonists such as ADP, thrombin and collagen. The defect is caused by mutations in one of the integrin genes, ITGA2 or ITGB3, encoding the αIIbβ3 receptor complex. Lack of expression or qualitative defects in αIIbβ3 results in a disturbed interaction between activated platelets and adhesive glycoproteins (fibrinogen at low shear and vWF at high shear) that bridge adjacent platelets during platelet aggregation (Fig. 1a). The Bernard-Soulier syndrome (MIM 231200) is caused by abnormalities in the GP Ib/IX/V receptor complex due to mutations in the genes for GPIbα, GPIbβ or GPIX (but there are no reports of BSS affecting the GPV gene) [22]. It is an autosomal recessive disorder with moderate to severe macrothrombocytopenia, decreased platelet survival and often a spontaneous bleeding tendency. The bleeding events can be severe but are usually controlled by platelet transfusion. Most heterozygotes, with a few exceptions, do not have a bleeding diathesis. BBS platelets aggregate normally in response to physiological agonists (ADP and collagen), have a weak response towards low concentrations of thrombin and do not agglutinate when platelet rich plasma is stirred with ristocetin or botrocetin [22].
Defects in G protein signaling resulting in an isolated platelet defect [36] are expected to be caused by a mutant G protein-coupled receptor (GPCR) since these can be cell-specific while the G proteins and their downstream effectors in this pathway are ubiquitously expressed. A dominantly inherited mutation (Arg60Leu) in the Gq-coupled TXA2 receptor was described in patients with a mild bleeding disorder characterized by defective platelet aggregation responses to TXA2 and its analogues (MIM 188070) [15]. In cultured cells, the Arg60Leu mutant was shown to impair phospholipase C (PLC) activation. Patients can be heterozygous (with some PLC activation left) or homozygous (without PLC activation) for this mutation but all have a life-long history of mucosal bleeding and easy bruising but no episodes of major bleeding such as hematuria, gastrointestinal bleeding or hemarthrosis [14]. The Gi-coupled ADP receptor P2Y12 (Fig. 1b) is responsible for the sustained, full aggregation response to ADP. P2Y12 deficiency (MIM 609821) is an autosomal recessive bleeding disorder characterized by excessive bleeding, prolonged bleeding time and abnormalities that are very similar to those observed in patients with secretion defects (reversible aggregation in response to weak agonists and impaired aggregation towards low concentrations of collagen and thrombin), except for the severely impaired response to ADP [2]. Study of the heterozygous P2Y12 defect revealed platelets that undergo a normal first wave of ADP induced aggregation but abnormal ATP secretion with different agonists [3, 42].
A defective platelet secretion is described for patients with absent alpha granules (gray platelet syndrome) or abnormal dense granules (delta-storage pool disease, δ-SPD) [37]. Gray platelet syndrome or α-SPD (MIM 139090) owes its name to the fact that the typically enlarged platelets devoid of α-granule staining, present with a gray color in a Wright-stained blood smear [32]. Most cases are sporadic though some family studies suggest an autosomal dominant inheritance. Affected members have a life-long history of mucocutaneous bleeding, which may vary from mild to moderate in severity, prolonged bleeding time, mild thrombocytopenia, abnormally large platelets and an isolated reduction of the platelet α-granule content. The molecular defect(s) in α-SPD have not yet been defined and further insights into the molecular mechanisms responsible for platelet exocytosis (as the SNARE proteins) will help in the search for causes of human platelets secretory disorders. δ-SPD (MIM 185050) may present as an isolated platelet function defect or can be associated with a variety of other congenital defects (see further). δ-SPD is characterized by a bleeding diathesis of variable degree, mildly to moderately prolonged skin bleeding time (fully related to the amount of ADP or serotonin contained in the granule), abnormal platelet secretion induced by several agonists and a reduced platelet aggregation. The δ-SPD platelets have decreased levels of the dense granule contents: ATP and ADP, serotonin, calcium and pyrophosphate (Fig. 1c). It was estimated that 10–18% of patients with a congenital abnormality of the platelet function have δ-SPD [12]. The inheritance pattern is autosomal recessive in some families while autosomal dominant in others but the molecular players responsible for δ-SPD are still unknown.
Human disorders comprising a platelet defect
Defects in platelet adhesion and subsequent platelet activation can also be due to an alteration in the platelet cytoskeletal organization, which consists of the microtubules and F-actin coupled to myosin, filamin and dystrophin [29]. Mutations in these widely expressed proteins result in a broader clinical phenotype. May-Hegglin anomaly (MIM 155100), Fechtner syndrome (MIM 153640), Epstein syndrome (MIM 153650), and Sebastian syndrome (MIM 605249) are characterized by macrothrombocytopenia, with or without different types of leukocyte inclusions, which can only be differentiated by an accurate ultrastructural examination [38]. In addition, patients with Epstein or Fechtner syndrome also suffer from nephritis, deafness, and congenital cataracts. Recently it became obvious that Sebastian platelet syndrome, May-Hegglin anomaly, Fechtner and Epstein syndrome have mutations in the same gene MYH9, encoding the 224-kD nonmuscle myosin heavy chain 9 polypeptide [26]. This gene is expressed in platelets, monocytes, granulocytes, the kidney, the auditory system but also in a lot of other tissues. MYH9 deficiency results in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton [1, 6]. The cytoskeletal defect could also explain the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets in MYH9 deficiency. Why patients with May-Hegglin anomaly, Fechtner syndrome, Epstein syndrome and Sebastian platelet syndrome have different signs and symptoms in other tissues than their common defect in platelets still remains to be elucidated.
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease (MIM 310200) characterized by progressive degeneration of muscle resulting in early death from respiratory or cardiac failure. DMD is caused by mutations in the dystrophin gene, a 427-kDa membrane-associated cytoskeletal protein. Evidence for a role of dystrophin in platelets started with the observation that DMD patients tend to bleed more during spinal surgery for scoliosis than do patients during the same surgery with other underlying conditions [7]. Other C-terminal isoforms of dystrophin due to differential promoter usage and/or alternative splicing at the 3′-end of the gene have been identified in platelets (Dp71), the retina (Dp260) and in the peripheral (Dp116) and central nervous systems (Dp140). It is well established that platelets contain a complex membrane cytoskeleton that resembles, at least in part, the cytoskeleton found in muscle, but a role for dystrophin during platelet activation still remains to be clarified [18, 27]. Recent studies showed a role for dystrophin in normal controls during platelet spreading and adhesion by regulating the α2β1 receptor but this was not studied in DMD patients [4, 5]. Another study describes a normal platelet function in DMD patients and blames the selective defect of primary hemostasis in DMD to impaired vessel reactivity [43].
Patients with an abnormal signal transduction are a heterogeneous group combined of defects in platelet G protein-coupled receptors (GPCR), the G proteins, and their effectors. Due to an extreme complex regulation between those key components (Fig. 1b), the incidence of this class of defects is definitely underestimated and the underlying molecular defects for the signaling problems are still largely unknown. Platelet Gs activity is easily determined using the platelet aggregation-inhibition test which gives a value for the Gs activity based on the inhibition of platelet aggregation by the rapid generation of cAMP after incubation with different Gs agonists such as prostacyclin or prostaglandin (Fig. 1b). A congenital Gs hyperfunction syndrome was described in three patients of two unrelated families due to a paternally inherited functional polymorphism in the extra-large stimulatory G-protein gene (XLαs) and its overlapping cofactor ALEX [8]. This XLαs variant is associated with Gs hyperfunction in platelets, leading to an increased trauma-related bleeding tendency but is also accompanied by neurological problems and brachydactyly (MIM 139320). A subsequent study revealed eight additional patients who paternally inherited the same XLαs polymorphism presenting with platelet Gs hyperfunction, brachydactyly, increased alkaline phosphatase and neurological problems or growth deficiency [9, 17].
Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which both the pituitary adenylyl cyclase-activating polypeptide (PACAP) and the vasointestinal peptide (VIP) are specific agonists. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from multiple neurological (epilepsy, hypotonia, convulsions, mental retardation, tremor, psychotic, hyperactive behavior), gastro-intestinal (diarrhea, vomiting) and endocrinological (hypoplasia of the pituitary gland, hypogonadotropic hypogonadism) problems and have a pronounced bleeding tendency (MIM 102980) [10]. The basal cAMP level in the patients' platelets was strongly elevated, providing a basis for the strongly reduced platelet aggregation. The VPAC1 signalling pathway also mediates megakaryocyte maturation and platelet formation (unpublished results). Patients with PACAP overexpression have a mild thrombocytopenia, a normal platelet survival, relatively small platelets and their bone marrow examination reveals almost no mature megakaryocytes.
There exist two rare syndromic forms of the δ-SPD: the Hermansky-Pudlak syndrome (HPS) and the Chediak-Hygashi syndrome (CHS). HPS (MIM 203300) consists of several genetically different autosomal recessive disorders, which share the clinical manifestations of oculocutaneous albinism, bleeding, and lysosomal ceroid storage resulting from defects of multiple cytoplasmic organelles: melanosomes, platelet-dense granules, and lysosomes hypopigmentation [25, 36]. HPS can arise from mutations in at least eight different genes known to date (HSP1 to HSP8), all coding for proteins involved in the formation, trafficking or fusion of intracellular vesicles of the lysosomal lineage [44]. CHS (MIM 214500) is also an autosomal recessive disorder, characterized by variable degrees of oculocutaneous albinism, large peroxidase-positive cytoplasmic granules in hematopoietic and non-hematopoietic cells, δ-SPD, recurrent infections, neutropenia, and an accelerated chronic lymphohistiocytic infiltration phase. The only known CHS-causing gene, LYST, codes for a large protein of unknown function but it seems that CHS is a disease of vesicle trafficking [16]. Most CHS patients present in early childhood and die before the age of 7 years unless treated by bone marrow transplantation [21]. About 10–15% of patients exhibit a much milder clinical phenotype and survive to adulthood but develop progressive and often fatal neurological dysfunction.
Human disorders examined by functional and morphological platelet assays
Some mainly polygenetic disorders can also be studied by using platelets although the patients only present with a subtle subclinical platelet phenotype. It is not easy to define disorders such as diabetes type 2 or some neurological diseases according to a defective adhesion, G protein signalling and secretion since the platelet defect is not yet well studied and usually overlaps different pathways. This part of the review will only briefly focus on the use of platelet research in our understanding of neurological disorders. It has been known for years that certain cellular functions are very similar in platelets and in neurosecretory cells [33] but the link between functional platelet studies and neurological defects is novel. A platelet and a neuron contain both mitochondria and dense core vesicles in which transmitters (such as serotonin) are stored. Platelets release serotonin upon activation (Fig. 2c) and the neuronal membrane is facilitated by a calcium dependent excitation-excretion coupling mechanism. In addition, both platelets and neurons contain functional neurotransmitter and neuromodulator receptor sites on their outer membrane such as adrenoceptors, serotonin receptors and serotonin transporters. More recently it was shown that platelets also express GABA and glutamate receptors [24, 34]. Serotonin uptake and release by platelets and serotonin plasma levels have been quantified and found to be altered in patients with bipolar disorder, schizophrenia, depression, aggression, autism, migraine, etc. [19, 23, 28]. Many epidemiological studies try to link these changes in activity of the serotonin transporter or changes in the density and responsiveness of the serotonin 2A receptor and the alpha2 adrenoceptor on the platelet membrane of these patients with genetic polymorphism in the corresponding genes. As for other epidemiological studies solid evidence for any linkage is not obvious but it is irrefutable that platelet studies have been invaluable in enabling an insight to be gained into the role of serotonin in a number of psychiatric and neurological disease [39, 41]. Major advances are expected of platelet research for this field in the near future since it is now obvious that besides the serotonin pathway, which was only the tip of the iceberg, many other pathways are chaired between platelets and neurons as well as the many gene products responsible for the regulation of granule formation, transport, secretion and endocytosis.
Conclusions
Platelet research is an expanding field originally studying isolated thrombopathies caused by the imbalance between thrombosis and hemostasis but more recently being able to bring novel insights in our understanding of human pathology in other clinical disciplines such as neurology, endocrinology and metabolic diseases. Platelets are easily accessible cells, and different techniques are possible to study platelet function and morphology under basal and activated conditions. Defects in platelet adhesion, G protein signalling and secretion can result from mutations in platelet-specific genes leading to isolated thrombopathies or from mutations in widely expressed genes leading to broader clinical phenotype including a platelet defect. In addition to using platelet research for diagnostic purposes, these platelet functional and morphological studies can also be used for research aims. From the close collaboration between clinicians of different disciplines, geneticists and the functional platelet research unit, novel insights in the pathogenesis of different human disorders are to be expected in the near future by using this strategy.

platelets
adhesion
secretion
g protein signalling
cytoskeleton