Platelet dense granule secretion in adolescents with conduct disorder and substance abuse: Preliminary evidence for variation in signal transduction

Platelet Dense Granule Secretion in Adolescents with Conduct Disorder and Substance Abuse: Preliminary Evidence for Variation in Signal Transduction Howard B. Moss and Jeffrey K. Yao

Platelet aggregation responses to agonists have been employed as peripheral indices of the physiological responsiveness and density of neurotransmitter receptors, and in investigations of membrane functioning in psychopathological conditions. In particular, there are mechanistic similarities between neuronal secretory and receptor dynamics, and those involved in platelet dense granule secretion. Consequently, we have explored the platelet dense granule secretory responses to various agonists in abstinent male adolescents who meet current psychiatric diagnostic criteria for Conduct Disorder and Psychoactive Substance Use Disorder (CD+/PSUD+) in contrast to controls ( C D - / P S U D - ) . The results showed a significant hyporesponsivity among experimental subjects to collagen, thrombin, adenosine diphosphate (ADP), ADP plus 0.2 p~gm of serotonin, and ADP plus 1.0 p~gm of serotonin. Only dense granule responses to arachidonic acid did not differentiate the groups. Taken together, the lack of agonist specificity suggests that a variation in signal transduction mechanisms could account for the observed reduction in dense granule secretion among CD+/PSUD+ adolescents. Association between dense granule secretory responses and substance use behavior, and comorbid psychiatric conditions are also examined. © 1996 Society of Biological Psychiatry Key Words: Substance abuse, conduct disorder, adolescents, platelets, signal transduction B I O L PSYCHIATRY

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Introduction For nearly two decades, the human platelet has been utilized as a peripheral model of neuronal functioning. Platelets show similarities to serotonergic (5-HT) pre- and From the Center for Education and Drug Abuse Research, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Address reprint requests to Howard B. Moss, M.D., Center for Education and Drug Abuse Research, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, 3811 O'Hara SWeet, Pittsburgh, PA 15213. Received April 25, 1995; revised August 7, 1995.

© 1996 Society of Biological Psychiatry

postsynaptic membranes (Passenon 1968; Stahl 1977; Pletcher 1978; Paul et al 1981; Lesch et al 1993), and a-adrenergic catecholaminergic neurons (Sneddon 1973; Pletcher 1981; Colman 1990). The human platelet 5-HT uptake site has been demonstrated to be analogous to the human brain 5-HT transporter (Lesch et al 1993). Platelet aggregation responses to neurotransmitter agonists have been employed as peripheral indices of the physiological responsiveness and density of the 5-HT 2 serotonin receptor (De Clerk et al 1982; McBride et al 1987, 1989). In addition, platelet aggregation responses to various agonists 0006-3223/96/$15.00 SSDI 0006-3223(95)00521-8

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have been employed in studies of membrane functioning in schizophrenia and other psychoses (Boullin et al 1975; Hefez et al 1980; Whalley et al 1984, Yao et al 1994). Platelets are activated by a variety of compounds. These include platelet-derived substances (i.e., 5-HT, adenosine diphosphate (ADP), and thromboxane A 2 (metabolized from arachidonic acid), vasoactive hormones (i.e., epinephrine and vasopressin), connective tissue components (i.e., collagen), peptides (i.e., platelet activating factor, and plasma products such as thrombin and von Willebrand factor). Platelet activation by such agonists is characterized by several well-described features: adhesion, dense granular secretion of adenosine triphosphate (ATP), shape change, and aggregation (Peterson and Lapetina 1994). The fact that the same activation response is common to this diverse group of agonists suggests that this capacity is localized to the level of the plasma membrane, and not a function of agonist surface receptor specificity. The receptor coupling to membrane-associated guanine nucleotidebinding proteins (G-proteins) seems to be a common feature of platelet surface receptors (Rubin 1990; Peterson and Lapetina 1994). Cellular activation reactions common to all agonists are thought to include effects on adenylate cyclase, the stimulation of phospholipase activity, calcium mobilization, phosphorylation of regulatory proteins, formation of new messengers, and discharge of storage granules (Colman 1990; Rubin 1990). Thus, individual variations in the magnitude of activated platelet secretory responses to various agonists may reflect a range of biological heterogeneity anywhere from the more proximal receptor functioning to the distal mechanics of dense granule secretion. Importantly, there are clear mechanistic similarities between neuronal secretory and receptor dynamics and those involved in platelet dense granule secretion (Manji 1992). The etiology of psychoactive substance use disorders (PSUD) among adolescents is thought to be multifactorial with biological, psychological, and social factors interacting to determine a PSUD outcome (Brook et al 1990; Tarter et al 1985). Research from the Ontario Child Health Study investigation reveals that, controlling for Attention Deficit Hyperactivity Disorder (ADHD), the only syndroreal psychiatric disorder that predicts substance abuse in either early or late adolescence is Conduct Disorder (CD) (Boyle et al 1992, 1993). Unfortunately, little is known about the neurobiological factors underlying CD, although genetic, neuropsychological, and psychophysiological research point to a putative biological substrate. A recent report suggests that variations in signal transduction mediated through the differential expression of the stimulatory G-protein may provide a biological substrate for the liability to alcoholism (Wand et al 1994). Consequently, we have explored the platelet granular secretory responses

to various agonists in abstinent male adolescents who meet psychiatric diagnostic criteria for CD and PSUD in contrast to those platelet responses from normal control male adolescents. Differential mean responses to specific individual agonists would be consistent with receptor-mediated variation, while commonalities in response patterns across agonists could reflect the status of signal transduction mechanisms, calcium mobilization, protein phosphorylation, and other intracellular events. In addition, we have examined dose-dependent 5-HTinduced amplification of the ADP secretory response because previous research suggests that it may reflect the Bmax of platelet 5-HT 2 receptor (McBride et al 1987). This is of interest because prior studies of adults with Antisocial Personality Disorder (Moss et al 1990), all of whom had CD in adolescence, and studies of impulsive and aggressive adult substance abusers (Virkkunen and Linnoila 1993) have suggested altered serotonergic functioning.

Methods

Subjects Adolescents with both a PSUD and a CD diagnosis (n = 66) were recruited by the Center for Education and Drug Abuse Research (CEDAR) from an adolescent substance abuse treatment program, as well as from two programs of the local juvenile justice system. One of these juvenile justice programs was mandated for adolescents convicted of drug-related arrests, and another was for adolescents adjudicated for violent offenses and a previous arrest record. Community-dwelling control subjects (n = 40) were recruited using newspaper advertising, posters, public service announcements, and word-of-mouth. In an effort to accrue the most representative and broad-based control group as possible, multiple recruitment approaches were employed. Exclusionary criteria for all subjects included a past or present history of a psychotic or neurologic disorder, or a lull-scale IQ less than 80. The legal guardians of all subjects provided informed consent, while adolescents provided informed assent, and were paid for their participation. The research protocol was approved by the Institutional Review Board of University of Pittsburgh Medical Center. The demographic characteristics of the experimental and controls samples are summarized in Table 1. In that head-of-household socioeconomic status (SES) differed significantly between groups, its status as a covariate of platelet dense granule secretion was also evaluated.

Psychiatric Diagnostic Evaluation The adolescents were administered an expanded version of the K-SADS-E (Orvaschel et al 1982) to determine the

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Table 1. Demographic Characteristics of Subjects CD+/PSUD+ (n = 66)

Controls (n = 40)

16.06 _+ 0.70 9.21 _+ 1.14

15.80 ± 0.97 9.55 ± 0.88

Age (yrs. ± SD) Education (highest grade completed ± SD) Head of household socioeconomic statusa

over-represented in the experimental group, the effects of these conditions on dense granule secretion was also evaluated.

Measures of Recent Drug Use 33.97 _+ 15.18

47.33 ± 13.91

at = 4.41,p < 0.00l

lifetime presence of DSM-III-R psychiatric disorders including CD. Since the K-SADS does not assess for PSUD diagnoses, the appropriate section of the SCID (Spitzer et al 1987) was modified for use in this population. "Best estimate" consensus diagnoses were finalized at clinical diagnostic conferences using the boy's self-report diagnostic interview, as well as any additional relevant clinical, collateral, or judicial information. The rates of the most prevalent lifetime diagnoses are displayed in Table 2. The significance of between-group differences in rates of psychiatric diagnoses was determined by Chi-square with continuity correction when appropriate. Both Attention Deficit Hyperactivity Disorder (ADHD) and Oppositional Defiant Disorder (ODD) were found to be significantly

Two sources of data were used to characterize recent drug use behavior. First, the Drug Use Screening Inventory (DUSI) was administered (Tarter 1990). This self-report questionnaire is a multidimensional, pencil and paper, self-report questionnaire consisting of a brief drug involvement section and 149 dichotomous (yes/no) items. These items correspond to ten domains known to be associated with substance involvement and behavioral problems in adolescents. The substance use domain quantifies the frequency of monthly use during the past year of a broad spectrum of psychoactive substances ranging from alcohol, to chewing tobacco in an ordinal fashion. Second, upon arrival at the center, subjects provide a urine specimen which is then subjected to commercial immunoassay drug screening tests (EZ-Screen, Environmental Diagnostics, Inc., Burlington, NC) to evaluate for the presence of metabolites of cannabis, cocaine, opioids, barbiturates, and amphetamines. Generally, these immunoassay tests are thought to reflect drug use over the prior 72 hours.

Table 2. Lifetime Psychiatric and Substance Use Disorder Diagnoses CD+/PSUD+

Control

Statistics

66 (100%) 18 (27.3%) 10 (15.2%) 1 (1.5%) 2 (3.0%) 0 (0) 2 (3.0%) 2 (3.0%) 1 (1.5%) 4 (6.1%)

0 (0) 3 (7.5%) 0 (0) 0 (0) 0 (0) 1 (2.5%) 0 (0) 0 (0) 0 (0) 0 (0)

n.s. X 2 = 4.95, p < .05 X 2 = 5.04, p < .05 n.s. n.s. n.s. n.s. n.s. n.s. n.s.

36 (54.5%) 40 (60.6%) 20 (30.3%) 38 (57.6%) 1 (1.5%) 6 (9.1%) 4 (6.1%) I 0 (15.2%) 4 (6.1%) 3 (4.5%) 2 (3.0%) 1 (1.5%) 4 (6.1%) 10 (15.2%) 14 (21.2%) 4 (6.1%)

0 (0) 0 (0) 1 (2.5%) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2.5%) 0 (0)

X2 X2 X2 X2

Lifetime Psychiatric Disorder." Conduct disorder Attention deficit disorder Oppositional defiant disorder Dysthymic disorder Social phobia Simple phobia Overanxious disorder Separation anxiety Obsessive-compulsive disorder Adjustment disorder

Lifetime Substance Use Disorder: Alcohol abuse Alcohol dependence Cannabis abuse Cannabis dependence Cocaine abuse Cocaine dependence Hallucinogen abuse Hallucinogen dependence Inhalant abuse Inhalant dependence Opioid abuse Opioid dependence PCP abuse PCP dependence Nicotine dependence Substance abuse N.O.S.

= = = =

30.65, p < .001 36.40, p < .001 10.43, p < .001 33.44, p < .001 n.s. n.s. n.s. X 2 = 5.03, p < .05 n.s. n.s. n.s. n.s. n.s. X 2 = 5.03, p < .05 X 2 = 5.72, p < .05 n.s.

Platelet Dense Granule Secretion in Conduct Disorder

Subjects who tested positive on the drug screens were not permitted to participate in that day's research protocol, consequently no samples were obtained.

Assessment Procedure The procedures and results reported here are components of a 26-hour research protocol implemented at the Center for Education and Drug Abuse Research (CEDAR). The overarching objective is to employ a prospective paradigm to understand the biobehavioral vulnerability and etiologic pathways of substance abuse. Initial screenings were conducted over the telephone. Potential subjects and their parents (or guardians) were specifically instructed about the necessity to remain abstinent from all psychoactive drugs, as well as aspirin, acetaminophen, ibuprofen, valproate, and other non-steroidal anti-inflammatory drugs for at least two weeks prior to the scheduled center assessment. No enrolled subjects were under treatment with nenroleptics or antidepressant drugs. The boys slept at the center on the first night to become acclimated to the experimental environment. At 7:30 AM the next day, after a undergoing a 12-hour fast, venipunctures were performed to obtain whole blood for platelet aggregation analysis and other research procedures. Blood specimens were then chilled in crushed ice and transported to the Neuropharmacology laboratory within 30 minutes after the blood draw. The aggregation tests were initiated immediately after arrival. The complete aggregation study of a single sample was usually concluded within 2 hours. Only fresh (unstored) blood samples were assayed.

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response of platelet dense granule secretion in response to various agonists has been studied elsewhere (IngermanWojenski and Silver 1984; Feinman et al 1985; Reiss et al 1986). We employed concentrations of agonists that were previously established as those inducing a near maximal aggregation and release response in whole blood. Peak ATP release usually occurs just prior to the maximum extent of aggregation (Challen et al 1982). Thus, ATP secretion was measured at the time of maximal platelet aggregation. ATP release was recorded on an IBM-PC AT interfaced with AGGRO/LINK (Chrono-Log Corp., Havertown, PA).

Statistical Analysis After generation of descriptive statistics, between-group comparisons of ATP secretory responses were performed by Student's t tests. Given the exploratory nature of the study, Bonferroni correction for multiple comparisons was not employed due to its augmentation of risk for Type II error. The significance of serotonergic amplification of ADP-induced responses was evaluated by paired t tests. The association of platelet secretory responses with recent drug-use self-reports was evaluated by Spearman bivariate correlations. Bivariate correlations between the various measures of dense granule secretion and SES was performed. A multivariate analysis of variance (MANOVA) to examine independent effects of CD, ADHD, and ODD on platelet dense granule secretory responses was also performed.

Results Evaluation of Agonist-Induced Platelet Dense Granule Secretion Approximately 450 Ixl of the freshly drawn whole blood was diluted with 450 txl of saline in a cuvette containing a disposable silaconized magnetic stirrer. The sample was placed into the Chrono-Log Lumi-Aggregometer (model 500VS) at 37°C and spun at 1000 rpm. 100 Ixl of Luciferin (0.16 mg/ml) was added to each sample, and allowed to equilibrate. Luminescence of a 2-nmole scale was established by adding 5 ~1 of adenosine triphosphate (ATP) standard solution (2 nmol) to the first sample prepared as above. Platelet secretory responses were first tested using a thrombin solution (1 U/ml). The agonist-induced ATP secretion was measured by comparing the change in luminescence in this sample to the 2-nmol ATP standard. The remaining samples were then stimulated with a variety of weaker agonists, including collagen (2 txg/ml), arachidonic acid (0.5 mM), adenosine diphosphate (ADP) (5.0 txM), ADP plus a low dose of 5-HT (0.2 Ixg), and ADP plus a high dose of 5-HT (1.0 t~g). The dose-

Agonist-lnduced A TP Secretion As can be seen in Figure 1, platelets from C D + / P S U D + adolescents show significantly less ATP secretion in response to thrombin (t = 4.82, p < 0.001), collagen (t = 3.42,p < 0.005), ADP (t = 2.77,p < 0.01), A D P + 0.2 Ixg 5-HT (t = 3.31, p < 0.005), and ADP + 1.0 I.tg 5-HT (t = 2.17, p < 0.05) in comparison to platelets from control adolescents. There were no significant differences between experimental and control platelets in dense granule secretory responses to arachidonic acid (t = 0.99, n.s.). None of the measures of dense granule secretion correlated with SES. MANOVA revealed significant main effects of CD (F = 4.88, p < 0.001), but not for ADHD (F = 1.08, n.s.) or ODD (F = 0.03, n.s.).

Serotonin-Induced Amplification of Dense Granule Secretion For the 0.2 ~g 5-HT dose paired t tests revealed significant amplification of the ADP response in both experimental

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(mean paired difference = - 0 . 0 7 _ 0.026 SEM; t --- 2.73, p < 0.01) and control groups (mean paired difference = - 0 . 0 8 ± 0.03 SEM; t = 2.51,p < 0.05). The 1.0 Ixg 5-HT dose paired t tests, however, revealed significant amplification of the ADP response in the experimental group (mean paired difference = -0.11 ± 0.028 SEM; t = 3.91, p < 0.001), but only a trend for the control groups (mean paired difference = - 0 . 0 6 ± 0.03 SEM; t = 1.75, p < 0.1).

Association With Drug Use Behavior As previously noted, no subjects were included who tested positive for any illicit drug of abuse. Little drug use behavior was noted among the normal controls with the exception of relatively low levels of lifetime alcohol use. In the C D + / P S U D + group, Spearman correlational analysis revealed very modest associations between secretory responses to ADP only, and self-reports of frequency of use of alcohol (r = 0.22, p < 0.08), use of cocaine (r = 0.24, p = 0.06), use of LSD (r = 0.29, p = 0.02), use of stimulants (r = 0.23, p = 0.07), and use of marijuana (r = 0.24, p = 0.06). It is noteworthy that these weak associations are of similar magnitude accounting for a range of 4.8% to 8.4% of the variance. Correlations between specific drug use reports and thrombin, collagen, arachidonic acid, and 5-HT enhanced ADP dense granule responses were highly nonsignificant.

Discussion Diminished dense granule secretory responses were observed in platelets from C D + / P S U D + boys in comparison to platelets from control youth across a range of agonists. Strong agonists, such as thrombin, produced large differential effects while weaker agonists such as ADP produced a smaller effect. The observation of a significant between-group effect in the same direction across multiple agonist treatments suggests that surface receptor specificity is not critical in determining this differential effect. Given the common signal transduction and intracellular second messenger processes associated with dense granule secretion across most of the receptor types examined in this study, the findings provide preliminary evidence for a variation in this intracellular processes in youth with C D + and PSUD. Only the platelet responses to arachidonic acid did not differentiate between the groups. It is noteworthy that the arachidonic acid platelet response differs significantly from other agonistinduced effects. Unlike other agonists for platelet activation, arachidonic acid must first be metabolized by two enzymatic steps (a cyclooxygenase reaction followed by thromboxane synthetase) to form thromboxane A 2 which

H.B. Moss and J.K. Yao

then binds to receptor sites and activates the platelet. One may speculate that the absence of between group differences is a result of the availability of a circulating pool of thromboxane A 2, contamination due to increased arachidonic acid availability as a consequence of marked erythrocyte lysis, or altered enzyme kinetics, however, this awaits empirical investigation. The frequent comorbidity of CD with ADHD and ODD is well-documented in the literature (Biederman et al 199 l). We evaluated whether or not the observed secretory variations were due to CD or its frequent psychiatric concomitants. The results support a robust effect of CD, but not for the presence of ADHD or ODD. It is unclear whether this diminished platelet secretory response is secondary to exposure to drugs of abuse or the trait of CD. Our analyses suggests a very weak association between only the ADP response and self reports of drug use. Responses to other strong and weak agonists did not even marginally correlate with drug use, however, self reports of drug use in a CD and juvenile justice population may be unreliable. In this investigation, no subjects were permitted to participate if their urine drug screen indicated drug use during the prior 72 hours, however, the life-span of the human platelet is 7-10 days (Nathan 1988), thereby providing a window of opportunity for physiologic alteration. Regrettably, urine drug screens do not assess for recent ethanol use, and we can only rely upon self-report data. The planned accrual of sample of non-CD, but PSUD+ adolescents may clarify this issue. Our investigation of 5-HT amplification of ADP platelet responses revealed significant amplification for the lower dose of 5-HT (0.2 Ixg) in both experimental and control groups, however, the higher dose of 5-HT (1.0 txg) produced statistically significant amplification in the C D + / P S U D + group only. Given prior research demonstrating a direct association between the Bmax of platelet 5-HT 2 receptor and the magnitude of amplification (McBride et al 1987), these data provide modest support for the hypothesis of an up-regulation of platelet 5-HT2 receptors in the C D + / P S U D + group possibly due to a serotonergic deficit. Alternatively, these differences could simply reflect an underlying variation in signal transduction. As previously noted, CD in childhood or adolescence is a necessary prerequisite for adult ASP in DSM-III-R and DSM-IV (APA 1994). Eysenck (1977) has argued that adult antisocial criminals have an underaroused central nervous system. A convergent literature documents psychophysiological under-responsivity to a variety of anticipated environmental stressors in ASP adults (Hare 1970, Fowles 1980, Ogloff and Wong 1990). ASP men also have demonstrated diminished adrenergic reactivity to anticipated stress (Lidberg et al 1978), as well as reduced

Platelet Dense Granule Secretion in Conduct Disorder

neuroendocrine response to pharmacological challenge with a serotonin agonist (Moss et al 1990). Similarly, we have recently reported a diminished salivary cortisol response to anticipated stress in impulsive, aggressive and delinquent prepubertal boys (Moss et al, 1995). A blunted cortisol response was also found to be associated with the magnitude of the father's childhood history of conduct disorder symptoms (Vanyukov et al 1993). A signal transduction and second messenger variation in CD, as suggested by these platelet findings, could provide a common biochemical mechanism of action for these multiple observations of hyporesponsivity in the antisocial

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spectrum of disorders. Investigations of the impact of drugs of abuse on this biochemical system and the psychosocial consequences of such hyporesponsivity may enhance our understanding of the associations between antisociality and the liability for substance use disorders. Such work is currently in progress.

This work was under the auspices of the Center for Education and Drug Abuse Research (a consortium of the University of Pittsburgh and St. Francis Medical Center) and supported by the National Institute on Drug Abuse (P50-DA 05605).

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