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Platelet dense granule secretion and aggregation in adolescents with conduct disorder: effects of marijuana use
Platelet Dense Granule Secretion and Aggregation in Adolescents with Conduct Disorder: Effects of Marijuana Use Howard B. Moss, Jeffrey K. Yao, and Kevin Lynch Background: We had previously reported a decrease in agonist-induced platelet dense granule secretion in blood samples from male adolescents with and without Conduct Disorder (CD). In an augmented sample, we have now employed multivariate modeling to examine the simultaneous effects of CD and regular monthly alcohol and marijuana use on both the dense granule secretion and aggregation phases of agonist-induced platelet responses. Methods: Blood samples were obtained from adolescents with and without a CD diagnosis. Platelet dense granule secretion and aggregation responses to a variety of agonists were examined in the laboratory. Results: Significant multivariate interactions of CD status with regular marijuana use were found for responses to collagen, ADP alone, and ADP plus 0.2 g. of serotonin. Responses in platelets from youth with CD, but without regular marijuana use differed from other subjects. Multivariate main effects of marijuana use alone on platelet responses to arachidonic acid and ADP plus 1.0 g. of serotonin were found. No effects of alcohol use were found. Conclusions: The results demonstrate an interaction between CD and the effects of chronic marijuana use for several agonists in this platelet model system, and further support the possibility of a variation in signal transduction mechanisms in CD. Biol Psychiatry 1999;46:790 –798 © 1999 Society of Biological Psychiatry Key Words: Conduct disorder, marijuana, platelet, signal transduction
Introduction
P
latelets are activated by a variety of compounds. These include platelet-derived substances such as 5-HT, adenosine diphosphate (ADP), and thromboxane A2 (meFrom the Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (HBM, JKY, KL). Address reprint requests to H.B. Moss, Department of Psychiatry, Temple University School of Medicine, 8th Floor, Jones Hall, 3401 N. Broad Street, Philadelphia, PA 19140. Received November 30, 1998; revised February 1, 1999; revised April 26, 1999; accepted April 27, 1999.
© 1999 Society of Biological Psychiatry
tabolized from arachidonic acid), vasoactive hormones such as epinephrine and vasopressin, connective tissue components such as collagen, peptides such as platelet activating factor, and plasma products such as thrombin and von Willebrand factor. Several well-described steps characterize platelet activation by such agonists: platelet adhesion, dense granular secretion of adenosine triphosphate (ATP), shape change, and finally aggregation (Peterson and Lapetina 1994). Receptors coupling to membrane-associated guanine nucleotide-binding proteins (G-proteins) seem 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 adenylyl 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). We had previously reported preliminary evidence of a diminution in the dense granule secretion of ATP in platelets obtained from a modest sample of abstinent adolescent polysubstance-abusing males with Conduct Disorder in response to treatment with collagen, thrombin, ADP, and ADP plus serotonin (Moss and Yao 1996). We speculated that because this observation of diminished responsivity was independent of the agonist used, the source of such variation might be at the level of cellular signal transduction mechanisms. In that report, effects of comorbid psychopathology were examined and simple bivariate correlations were employed to examine any putative effects of substance use behavior on platelet dense granule secretory responses yielding equivocal results. In this report, we have substantially increased our sample size of adolescents with and without Conduct Disorder who display a wider range of substance use 0006-3223/99/$20.00 PII S0006-3223(99)–112-2
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behavior beyond the history of polysubstance abuse characterizing our original sample. This expanded sample has permitted more informative analyses, and provided the opportunity to directly model the effects of regular use of two important “gateway” drugs, alcohol and marijuana, simultaneously with the platelet effects of Conduct Disorder (CD). These two drugs are of interest as potential mediators of the effect of CD disorder on platelet functioning because substance use behavior and disorders are over-represented among CD adolescents. Alcohol use behavior of adolescents could explain the effect of CD because, to date, at least eight studies have demonstrated reduced agonist-induced platelet aggregation in adult alcoholics entering treatment (reviewed in Rubin and Rand 1994). Similarly, cannabinoids, the active ingredients in marijuana have been shown to have inhibitory effects on agonist-induced platelet aggregation (Formukong et al 1989). As a further extension of the original report, we have also analyzed herein both the aggregation responses of platelets treated with a battery of agonists and their respective dense granule secretory responses. This permits the evaluation of two distinct stages of the aggregation process within a multivariate model framework. Our working hypothesis was that platelets from abstinent CD adolescents would demonstrate diminished platelet dense granule secretory and aggregation responses explicable by alcohol, or marijuana use behavior. Surprisingly, the results suggest an unexpected and complex relationship between CD and marijuana use alone on this platelet model system.
Methods and Materials Subjects Male adolescents with a CD diagnosis (CD⫹; n ⫽ 102) and male adolescents without a CD diagnosis (CD⫺; n ⫽ 101) were recruited by the Center for Education and Drug Abuse Research (CEDAR) from adolescent substance abuse treatment programs, the local juvenile justice system, and from the community. The mean age for the CD⫹ adolescents (⫾SD) was 16.01 ⫾ 0.69 years and the mean age (⫾SD) for the CD-adolescents was 15.49 ⫾ 0.65 years. Community-dwelling subjects were recruited using newspaper advertising, posters, public service announcements, and word-of-mouth. Several of these subjects were part of a longitudinal cohort initially ascertained at age 10 and followed into adolescence. Multiple recruitment approaches were employed in an effort to accrue as representative and broad-based a sample as possible as possible. Exclusionary criteria for all subjects included a past or present history of a psychotic or neurologic disorder, or a full-scale IQ less than 80. The legal guardians of all subjects provided informed consent, whereas adolescents provided informed assent, and were paid for their participation. The Institutional Review Board of University of Pittsburgh Medical Center approved the research protocol.
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Psychiatric Diagnostic Evaluation The adolescents were administered an expanded version of the K-SADS-E (Orvaschel et al 1982) to determine the lifetime presence of DSM-III-R psychiatric disorders including CD. In that the K-SADS does not assess for substance use disorder 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 subject’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 1. The significance of between-group differences in rates of psychiatric diagnoses was determined by Chi-square with continuity correction. In that both alcohol use disorders and cannabis use disorders were also over-represented in CD⫹ youth, more direct measures of the use of these drugs were also examined within the same models.
Measures of Recent Drug Use Two sources of data were used to characterize recent drug use behavior. First, the revised Drug Use Screening Inventory (DUSI-R) 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 10 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 that is then subjected to commercial immunoassay drug screening tests (EZ-Screen, Environmental Diagnostics, Inc.) 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. Subjects who tested positive on the drug screens were not permitted to participate in that day’s research protocol, and were required to return after a week of abstinence. Blood samples were obtained only after a negative drug screen. This effort was designed to eliminate potential confounds of variations in recency of substance use on the biological phenomena of interest.
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 over-arching 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 2 weeks before the scheduled center assessment. No
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Table 1. Common Lifetime Psychiatric Disorders among Conduct Disordered (CD⫹) and Nonconduct Disordered (CD⫺) Adolescents
DSM-III-R disorders:
CD⫹ (n ⫽ 102)
CD⫺ (n ⫽ 101)
Attention deficit hyperactivity disorder
24 (23.5%)
2 (2.0%)
Oppositional defiant disorder
12 (11.8%)
4 (4.0%)
Major depressive disorder
14 (13.7%)
3 (3.0%)
Simple phobias
0 (0%)
2 (2.0%)
Social phobia
2 (1.9%)
0 (0%)
Adjustment disorder
5 (4.9%)
1 (1.0%)
Alcohol use disorder (abuse or dependence)
71 (69.6%)
5 (5.0%)
Cannabis use disorder (abuse or dependence)
65 (63.7%)
4 (4.0%)
Nicotine dependence
14 (13.7%)
3 (3.0%)
8 (7.8%)
2 (2.0%)
12 (11.8%)
1 (1.0%)
Cocaine use disorder (abuse or dependence) Hallucinogen use disorder (abuse or dependence) Inhalant use disorder (abuse or dependence)
8 (7.8%)
enrolled subjects were under treatment with neuroleptics 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 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 blood samples were assayed.
Evaluation of Agonist-induced Platelet Dense Granule Secretion and Aggregation Approximately 450 l of the freshly drawn whole blood was diluted with 450 l of saline in a cuvette containing a disposable siliconized magnetic stirrer. The sample was placed into the Chrono-Log Lumi-Aggregometer (model 500VS) at 37°C and spun at 1000 rpm. To each sample, 100 l of Luciferin (0.16 mg/ml) was added and allowed to equilibrate. Luminescence of a 2-nmol scale as established by adding 5 l 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
0 (0%)
Chi-square (2) (continuity-corrected); df ⫽ 1 2 ⫽ 19.22 p ⬍ .001 2 ⫽ 3.25 p ⫽ .07 (trend) 2 ⫽ 6.31 p ⬍ .01 2 ⫽ 0.52 p ⫽ NS 2 ⫽ 0.48 p ⫽ NS 2 ⫽ 1.52 p ⫽ NS 2 ⫽ 87.84 p ⬍ .001 2 ⫽ 78.15 p ⬍ .001 2 ⫽ 6.31 p ⬍ .05 2 ⫽ 2.58 p ⫽ NS 2 ⫽ 8.11 p ⬍ .005 2 ⫽ 6.31 p ⬍ .05
samples were then stimulated with a variety of weaker agonists, including collagen (2 g/ml), arachidonic acid (0.5 mM), adenosine diphosphate (ADP) (5.0 M), ADP plus a low dose of 5-HT (0.2 g), and ADP plus a high dose of 5-HT (1.0 g). The dose-response of platelet dense granule secretion in response to various agonists has been studied elsewhere (Ingerman-Wojenski 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 before the maximum extent of aggregation (Challen et al 1982). Thus, ATP secretion and aggregation were measured at the time of maximal platelet aggregation for all agonists except thrombin. Platelet aggregation was simultaneously quantified by measuring the change in impedance of the sample against the aggregometer’s internal 20 Ohm standard. Both ATP secretion and impedance changes were measured with an IBM-PC interfaced with AGGRO-LINK (Chrono-Log Corp.) and recorded on a HewlettPackard PaintJet printer.
Statistical Analysis Alcohol and cannabis (marijuana) use information from the DUSI was re-coded into variables indicating either that the subject has “never used” the drug, “uses the drug less than 10 times a month,” and “uses more than 10 times a month.” These responses grouped according to CD status are displayed in Table 2. Although tobacco use behavior was deemed relevant to the
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Table 2. Relationship between Conduct Disorder and Alcohol and Marijuana Use Behavior Self-Reported by Adolescents in the Study
Alcohol use behaviora Never use Regular monthly use Marijuana use behaviorb Never use Regular monthly use
CD⫹ (n ⫽ 102)
CD⫺ (n ⫽ 101)
43 (42.2%) 59 (57.8%)
71 (70.3%) 30 (29.7%)
50 (49%) 52 (51%)
74 (73.3%) 27 (26.7%)
⫽ 16.32, df ⫽ 1, p ⬍ .0001. ⫽ 12.55, df ⫽ 1, p ⬍ .0001.
a 2 b 2
research questions herein, a selective pattern of missing data on this variable precluded its use in these analyses. The measures of alcohol and marijuana use behavior were then dichotomized into a variable indicating either “no use” of the drug or “regular monthly use” to increase factor size and enhance statistical power. A multivariate general linear model approach was employed to simultaneously test the effects of the presence or absence of CD, regular alcohol and regular marijuana use behavior on paired agonist-specific secretory ATP responses and aggregation-associated impedance responses. The distributional characteristics of each agonist-specific secretory ATP response and aggregationassociated impedance change were evaluated for deviation from normality. Logarithmic transformations were employed to reduce skewness for all dense granule secretion and aggregation dependent variables. After the recommendations of Tabachnick and Fidell (1996), we examined the following characteristics for each of the selected models: (a) univariate or multivariate within-cell outlier, (b) normality of dependent variables within cells, (c) homogeneity of variance-covariance matrices within cells, and (d) linearity of the independent-dependent variable relationships within cells. After these adjustments to the variables, their characteristics and relationships were found to be without significant violations of the assumptions noted above. In some cases, there were unequal distributions of participants among variable categories. The distribution of values in cells tended to increase with cell count, that would tend to cause more conservative results (Tabachnick and Fidell 1996). Initially, both main effects and interactions were examined. Non-significant interaction terms were removed from the final models. For significant models, and for thrombin secretory responses alone, univariate F-tests were performed to examine individually the secretory and impedance responses. These analyses were conducted in SPSS for Windows 8.0 (SPSS Inc., Chicago, Ill.) as implemented on a personal computer.
Results Figure 1 demonstrates the relative positions of each of the paired platelet secretory and aggregation responses grouped according to agonist, CD status and marijuana use status essentially summarizing the multivariate analyses.
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As previously noted, only dense granule secretory responses were examined for thrombin, consequently thrombin data are not shown in this graphic representation. As can be seen in the variations in shape and position, different response patterns may be observed for the collagen, arachidonic acid, ADP and the ADP plus serotonin treatments. Collagen and arachidonic acid produce the most robust responses in both the dense granule secretory phase and the aggregation phase. The addition of serotonin shifts the ADP responses to the right along the horizontal axis indicating an augmentation of the aggregation phase, but producing little variation in the dense granule secretion phase. Furthermore, the qualitative effects of CD and marijuana use status differ within each agonist system investigated. Table 3 displays the dense granule secretory and aggregation responses to each of the agonists employed. For thrombin-induced dense granule secretion, a significant interaction of CD status with marijuana use was found (F1,194 ⫽ 4.46, p ⬍ .05), such that platelets from CD⫹ adolescents without regular marijuana use a diminished ATP secretory response compared to CD⫹ regular marijuana users and CD⫺ adolescents. Effects of alcohol use were non-significant. For platelet responses to collagen, a significant interaction of CD status and marijuana use status was found together on the secretory and aggregation responses (F2,194 ⫽ 4.56, p ⬍ 05). For CD⫹ adolescents who were not regular users of marijuana, their platelet secretory responses were less than CD⫹ regular users, but their aggregation responses were greater. For CD⫺ adolescents, regular marijuana users had somewhat greater dense granule secretory response and a greater aggregation response than nonusers of marijuana. Post-hoc univariate F-tests revealed that the interaction of CD by marijuana usage was most pronounced on the aggregation phase of the response to collagen (F1,195 ⫽ 7.67, p ⬍ .01), not the dense granule secretory responses (F1,195 ⫽ 0.84, p ⫽ NS). No effects of alcohol use behavior were noted. For arachidonic acid-induced dense granule secretory and aggregation responses, there was a main effect of regular marijuana usage (F2,169 ⫽ 3.18, p ⬍ .05). Univariate analyses revealed that this effect was predominantly impacting upon platelet aggregation (F1,170 ⫽ 3.42, p ⫽ .06) such that regular marijuana users showed a diminished aggregation response compared to nonusers. No significant univariate effects of marijuana usage upon dense granule secretion were observed (F1,170 ⫽2.17, p ⫽ NS). No effects of alcohol usage were observed. For platelet responses to adenosine diphosphate (ADP), significant interactions of CD status with marijuana use status on the conjoint platelet dense granule and aggregation responses were observed (F2,146 ⫽ 3.62, p ⬍ .05). For
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Figure 1. Relative positions of paired platelet dense granule secretion and aggregation mean values grouped according to Conduct Disorder status (CD⫹ or CD⫺) and regular marijuana use status (MJ⫹ or MJ⫺) for each agonist. The varying shapes of the polygons for collagen, arachidonic acid, ADP and ADP plus 5-HT responses demonstrate the heterogeneity of responses of these biological systems. Collagen and arachidonic acid appear as “strong” agonists, whereas ADP and ADP plus serotonin are “weaker” agonists. The addition of serotonin (5-HT) to ADP shifts responses somewhat to the right primarily along the horizontal axis indicating a predominant effect on aggregation, not dense granule secretion. Significant interactions of CD with marijuana use were found for collagen (p ⬍ .05), ADP (p ⬍ .05), and ADP plus 0.2 g 5-HT (p ⬍ .05). For ADP, and ADP plus 5-HT, the CD⫹/MJ⫺ group shows a lesser agonist response for the dense granule secretory and aggregation phases. For collagen, the CD⫹/MJ⫺ group seems to have a greater aggregation response, but a lessor dense granule secretory response. Main effects of marijuana use were found for arachidonic acid (p ⬍ .005), and ADP plus 1.0 g 5-HT (p ⬍ .05) such that platelets from MJ⫹ subjects have reduced aggregation responses.
CD⫹ adolescents, regular marijuana users had heightened dense granule secretory responses and aggregation responses compared to CD⫹ nonusers of marijuana. For CD⫺ adolescents, marijuana users did not differ from nonusers in terms of dense granule responses, but users had a slightly greater aggregation response than nonusers of marijuana. Post-hoc univariate analyses revealed that interaction between CD and marijuana use status was mostly due to the effects on dense granule secretory responses (F1,147 ⫽ 4.18, p ⬍ .05) and not on aggregation responses (F1,147 ⫽ .00, p ⫽ NS). Effects of alcohol use behavior were not significant. For the platelet responses to ADP plus 0.2 g of serotonin, significant interactions between CD and marijuana use status were also found (F2,156 ⫽ 4.00, p ⬍ .05).
For platelets from CD⫹ adolescents, marijuana users showed an increased dense granule secretory response, but a lessor degree of aggregation than CD⫹ nonusers of marijuana. For platelets from CD⫺ youth, marijuana users had a lesser dense granule secretory response, and reduced aggregation response than nonusers of marijuana. The effect of alcohol use behavior was not significant. For the ADP plus 1.0 g serotonin condition, there was only an effect of marijuana use status observed (F2,154 ⫽ 4.59, p ⬍ .05). Regular marijuana users had somewhat increased dense granule secretion but univariate analysis revealed a significantly reduced aggregation (F1,155 ⫽ 3.70, p ⫽ .06). No effect of alcohol use behavior was observed. Several patterns were also observable in the data in
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Table 3. Mean (⫾SEM) Dense Granule Secretion and Aggregation in Platelets from Adolescent Males with and without Conduct Disorder and Regular Monthly Marijuana Use Conduct disorder (n ⫽ 102) Regular marijuana use (n ⫽ 52)
Agonist Thrombina Collagenb Arachidonic acidc Adenosine diphosphate (ADP)d ADP ⫹ 0.2 g 5-HTe ADP ⫹ 1.0 g 5-HTf
Dense granule secretion (mol ATP)
No conduct disorder (n ⫽ 101)
No marijuana use (n ⫽ 50)
Aggregation (ohms)
Dense granule secretion (mol ATP)
1.92 ⫾ 0.09 1.21 ⫾ 0.06 0.93 ⫾ 0.06 0.51 ⫾ 0.03
NA 29.55 ⫾ 1.33 23.65 ⫾ 1.79 11.45 ⫾ 1.73
0.53 ⫾ 0.06 0.54 ⫾ 0.06
15.58 ⫾ 1.73 18.72 ⫾ 1.92
Regular marijuana use (n ⫽ 27)
No marijuana use (n ⫽ 74)
Aggregation (ohms)
Dense granule secretion (mol ATP)
Aggregation (ohms)
Dense granule secretion (mol ATP)
1.59 ⫾ 0.07 1.06 ⫾ 0.08 0.76 ⫾ 0.06 0.28 ⫾ 0.03
NA 36.66 ⫾ 1.48 27.79 ⫾ 1.97 8.61 ⫾ 1.50
1.96 ⫾ 0.10 1.14 ⫾ 0.07 0.92 ⫾ 0.09 0.54 ⫾ 0.06
NA 30.15 ⫾ 1.71 25.00 ⫾ 1.96 15.58 ⫾ 2.08
1.99 ⫾ 0.07 NA 1.09 ⫾ 0.05 28.93 ⫾ 0.96 0.90 ⫾ 0.05 26.03 ⫾ 1.33 0.54 ⫾ 0.05 14.36 ⫾ 1.38
0.37 ⫾ 0.04 0.39 ⫾ 0.04
19.64 ⫾ 1.96 20.42 ⫾ 1.92
0.54 ⫾ 0.05 0.50 ⫾ 0.06
17.78 ⫾ 2.01 20.26 ⫾ 1.97
0.59 ⫾ 0.05 20.05 ⫾ 1.15 0.53 ⫾ 0.04 22.34 ⫾ 1.20
Aggregation (ohms)
Significant multivariate main effects and interactions: a CD ⫻ Marijuana: F 1,194 ⫽ 4.46, p ⬍ .05. b CD ⫻ Marijuana: F 2,194 ⫽ 4.56, p ⬍ .05. c Marijuana effect: F 2,169 ⫽ 3.18, p ⬍ .05. d CD ⫻ Marijuana: F 2,146 ⫽ 3.62, p ⬍ .05. e CD ⫻ Marijuana: F 2,156 ⫽ 4.00, p ⬍ .05. f Marijuana effect: F 2,154 ⫽ 4.59, p ⬍ .05.
Table 3. Generally, platelet dense granule secretion and aggregation responses across agonists tended to be lowest in the CD⫹ no regular marijuana use group. Platelet dense granule responses in the CD⫹ regular marijuana use group resembled those of the CD⫺ no marijuana use group.
Discussion This study extends, but does not replicate our original observation of a variation in platelet dense granule secretion in CD⫹ adolescent males with polysubstance abuse. In this enriched sample, we have employed multivariate techniques to model the simultaneous effects of CD, alcohol use behavior and marijuana use behavior on both phases of the platelet responses to several established agonists. These results suggest a much greater complexity in the determinations of both phases of platelet responses than assumed in the original report of polysubstance abusing CD⫹ males. Herein, the presence or absence of CD was found to significantly interact with marijuana use behavior in a manner that was fairly independent of the specific agonist system examined. Significant interactions between CD status and marijuana use status were found for responses to thrombin, collagen, ADP, and ADP plus 0.2 g of 5-HT. Thus, the impact of regular marijuana usage on platelet responsivity to these agonists depended upon whether the subject was either CD⫹ or CD⫺. To add further complexity to the problem, many of the effects of this interaction were observed to be more pronounced in either the dense granule secretory phase or the aggregation
phase of the response again depending upon the agonist system. The least pronounced platelet responses in both phases were found in samples from adolescents with CD, but who did not use marijuana. Curiously, with the exception of collagen responses, platelet responses from marijuana using, CD⫹ adolescents tended to resemble those from CD⫺, non-marijuana using subjects. The results suggest that platelets from CD⫹ youth, that do not regularly use marijuana, demonstrate a variation in responsivity, regardless of the identity of the agonist employed. Because platelet receptor coupling to membrane-associated guanine nucleotide-binding proteins (Gproteins) is a common feature of platelet surface receptors (Peterson and Lapetina 1994), differences in plasma membrane or G-protein function could account for the observed variations in platelet responses. In a recent 31P magnetic resonance spectroscopy study, we reported a regional reduction of phosphodiesters in the brains of prepubertal children with CD or Oppositional Disorder (Moss et al 1997). This reduction in the breakdown products of membrane phospholipids could reflect a more global variation in the composition of plasma membranes. On the other hand, Wand et al (1994) suggested that variations in signal transduction mediated through the differential expression of the stimulatory G-protein might provide a biological substrate for the familial liability to alcoholism. Cellular activation reactions common to most platelet agonist receptors include second messenger effects such as activation of adenylyl cyclase, the stimulation of phospholipase activity, calcium mobilization, the phosphoinositide cascade reaction, phosphorylation of regulatory proteins
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by kinases, the formation of new messengers and discharge of storage granules (Colman 1990; Rubin 1990). Biological variations due to CD could be localized to any of these intra-cellular processes. Investigations of these putative mechanisms await further research. Pine et al (1996) have reported platelet serotonergic variations such as reductions in the density of the platelet 5-HT2a receptor in delinquent adolescents. The magnitude of serotonin augmentation of ADP-induced platelet aggregation, as employed herein, has been demonstrated to reflect the density of platelet 5-HT2a receptors (McBride et al 1987). Consequently, if one examines only the 5-HT augmentation of the ADP platelet response, the results might imply a specific reduction in 5-HT2a receptors among adolescents with CD. The observation of this effect across multiple agonists lends less credence to the solitary interpretation. Parsimony suggests that the variations in platelet responses among CD⫹/MJ⫺ youth across multiple agonists are less likely to be based on individual effects localized to specific receptors. It is more probable that these multiple agonist effects are due to factors common to these varied receptors, either at the level of the biological membrane or intracellular signaling mechanisms. It is tempting to speculate that the biological substrate mediating the under-responsivity of platelets might also mediate other aspects of under-reactivity that has been hypothesized to be the basis for the development of deviant behavior, including conduct disorder and antisocial personality disorder. Eysenck (1977) has argued that adult antisocial criminals have an under-aroused 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 neuroendocrine response to pharmacological challenge with a serotonin agonist (Moss et al 1990). In addition, we have observed in children a hyporesponsivity of the hypothalamic-pituitary-adrenal axis to anticipated stress that was associated with higher conduct disorder symptom counts and paternal antisociality (Vanyukov et al 1993; Moss et al 1995; Moss et al 1999). Such under-reactivity may be either constitutive (reflect a higher genetic predisposition), or result from an adaptation to prolonged stress, perhaps caused by parental antisociality (e.g., high aggression) and substance abuse, or an interaction of the two. Pharmacological effects of marijuana on platelet responsivity are less obscure and better supported by clinical and pre-clinical investigation. Schaeffer et al (1979) reported a decrease in platelet aggregation among marijuana smokers. Formukong et al (1989) subsequently examined
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the effects of adding several cannabinoid constituents of marijuana to human and rabbit platelets and observing their activation in vitro. Olvetol, cannabidiol, cannabinol and tetrahydrocannabinol were all found to inhibit adrenaline-induced platelet aggregation in a dose-dependent fashion. Only olvetol inhibited phorbol ester-induced aggregation limiting the possibility that all cannabinoid effects are at the level of protein kinase C. The observation that cannabinoids were effective inhibitors of platelet activating factor-induced aggregation suggests that these effects may be on the prostaglandin cascade, perhaps through inhibition of cyclo-oxygenase. The method used by these investigators was different from that employed by us in this report. Formukong et al. used an aggregometer that was based on changes in light transmission produced by the clumping of platelets. Their results may be interpreted as being analogous to our impedance aggregation measure, but not our dense granule secretion measure. More recently, Skosnik, Yao and Park (unpublished data), using dense granule secretion and impedance aggregation measures, have shown that the addition of two endogenous cannabinoids to blood produced an inhibition of ADPinduced (but not collagen or arachidonic acid) platelet aggregation. Furthermore, they have noted a hyper-responsivity of platelets from schizophrenic patients compared to controls in terms of cannabinoid inhibition of aggregation demonstrating an interaction between psychopathology and the cannabis effects on this peripheral model system. Our data are not totally consistent with the in vitro studies, although some inhibition seems to be appreciable in terms of the second phase of aggregation for arachidonic acid responses and the ADP plus 1.0 g serotonin responses. The discrepancy between the in vitro studies this clinical investigation may be due to the fact that our subjects were required to be abstinent for 72 hours before blood sampling. The life-span of the human platelet is 7–10 days (Nathan 1988), thereby providing a window of opportunity for physiologic alteration by highly lipophilic cannabinoids. Thus, any effects noted herein may be attributable to chronic exposure to marijuana rather than acute effects of cannabinoids. It was admittedly surprising that we were not able to find an effect of alcohol use on platelet aggregation. There is a significant extant literature reporting the impact of alcoholism on platelet functioning in adults (Rubin and Rand 1994). Nonetheless, we found no effects of alcohol use behavior on any of the agonist systems studied, perhaps due to the differences in the alcohol use behaviors and clinical presentations of adolescent alcohol abuser compared to adult alcoholics. Adolescents typically binge drink, whereas daily intoxication is more frequently found among adult alcoholics. (Stewart and Brown 1995; Martin et al 1995). Thus, the short life span of the platelet,
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combined with more sporadic alcohol exposure, and the 72-hour abstinence requirement might have limited our ability to detect alcohol effects on platelet physiology. There are several important limitations in this study that impacts upon its interpretation and generalizability. First and foremost is the selectivity of the adolescent sample. The majority of CD cases were obtained through adolescent substance abuse treatment programs, and the local juvenile justice system. Thus, they may represent a more extreme variant due to Berksonian bias (Berkson 1946), and consequently have a greater probability of a constitutive difference from community-dwelling subjects. Second, our sample size is not sufficiently large to adequately model and test any platelet effects due to comorbid psychiatric disorders. As can be seen in Table 1, there is substantial psychiatric co-morbidity in the sample. Larger cell sizes for each comorbid combination would be necessary to permit informative multivariate analyses. In our previous report, we used univariate methods to rule out effects of attention deficit disorder, oppositional defiant disorder, and major depression (Moss and Yao 1996). Third, self-report information on drug use behavior, especially among conduct disturbed adolescents maybe be inaccurate due to response biases in this population. Fourth, our diagnoses are based upon DSM-III-R, as opposed to DSM-IV. Thus, we cannot accurately subtype our CD adolescents into childhood versus adolescent onset Conduct Disorder in accordance with DSM-IV criteria. Lastly, inconsistencies in the collection of data on cigarette smoking behavior (as opposed to the presence or absence of nicotine dependence) precluded an adequate examination of the effects of tobacco smoking on platelet aggregation responses. Because nicotine dependence is over-represented in the CD⫹ group, we cannot rule out effects of nicotine, tars, or carbon monoxide on platelet functioning. We hope to examine effects of smoking more directly employing a newly accrued sample of adolescents. Similarly, hallucinogen use disorders are over-represented in the CD⫹ group, but the cell size is not sufficiently large to permit meaningful analyses. This work was performed under the auspices of the Center for Education and Drug Abuse Research (a consortium of the University of Pittsburgh and St. Francis Medical Center) (P-50-AA 08746 and supported by the National Institute on Drug Abuse (P50-DA 05605; Dr. Moss DA-00308). Dr. Lynch is supported by National Institute on Alcohol Abuse and Alcoholism (P-50-AA 08746).
References Berkson J (1946): Limitations of the application of fourfold table analysis to hospital data. Biometrics 2:47–53. Challen A, Branch WJ, Cummings JH (1982): Quantitation of platelet mass during aggregation in the electronic (Wellcome)
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Whole Blood Aggregometer. J Pharmacol Methods 8:115– 122. Colman, RW (1990): Platelet receptors. Hematol Oncol Clin North Am 4:27– 42. Eysenck, HJ (1977): Crime and Personality, 3rd ed. London: Routledge and Kegan. Feinman R, Detwiler T, Ingerman-Wojenski C (1985). The lumi-aggregometer as a research tool and clinical tool. In: Longnecker G, editor. The Platelets: Physiology and Pharmacology, Orlando: Academic Press Inc., pp 429 – 440. Formukong EA, Evans AT, Evans FJ (1989): The inhibitory effects of cannabinoids, the active constituents of Cannabis sativa L. on human and rabbit platelet aggregation. J Pharm Pharmacol 41:705–709. Fowles, DC (1980): The three arousal model: implications of Gray’s two-factor learning theory for heart rate, electrodermal activity and psychopathy. Psychophysiology 17:87–104. Hare RD (1970): Psychopathy. Theory and research. New York: John Wiley & Sons. Ingerman-Wojenski CM, Silver MJ (1984): A quick method for screening platelet dysfunctions using the whole blood lumiaggregometer. Thromb Haemostas 51:154 –156. Lidberg L, Levander S, Schalling D, Lidberg Y (1978): Urinary catecholamines, stress, and psychopathy: a study of arrested men awaiting trial. Psychosom Med 40:116 –125. Manji HK (1992): G proteins: implications for psychiatry. Am J Psychiatry 149:746 –760. Martin CS, Kaczynski NA, Maisto SA, Bukstein OM, Moss HB (1995): Patterns of DSM-IV alcohol abuse and dependence symptoms in adolescent drinkers. J Stud Alcohol 56:672– 680. McBride PA, Mann JJ, Polley MJ, Wiley AJ, Sweeney JA (1987): Assessment of binding indices and physiological responsiveness of the 5-HT2 receptor on human platelets. Life Sci 40:1799 –1809. Moss HB, Talagala SL, Kirisci L (1997): [31P] Magnetic resonance brain spectroscopy of children at-risk for a substance use disorder: preliminary results. Psychiatry Res 76:101–112. Moss HB, Vanyukov MM, Martin CS (1995): Salivary cortisol responses and the risk for substance abuse in prepubertal boys. Biol Psychiatry 38:547–555. Moss HB, Vanyukov M, Yao JK, Kirillova GP (1999): Salivary cortisol responses in prepubertal boys: associations with exposure to paternal substance abuse and drug use behavior during adolescence. Biol Psychiatry 45:1293–1299. Moss HB, Yao JK (1996): Platelet dense granule secretion in adolescents with conduct disorder and substance abuse: preliminary evidence for variation in signal transduction. Biol Psychiatry 40:892– 898. Moss HB, Yao JK, Panzak GL (1990): Serotonergic responsivity and behavioral dimensions in antisocial personality disorder with substance abuse. Biol Psychiatry 28:325–38. Nathan DG (1988): Introduction: hematologic diseases. In: Wyngaarden JB, Smith LH, editors. Cecil Textbook of Medicine, Philadelphia: W.B. Saunders, pp 873– 878. Ogloff JR, Wong, S (1990): Electrodermal and cardiovascular evidence of a coping response in psychopaths. Criminal Justice Behav 17:231–245. Orvaschel H, Puig-Antich J, Chambers W, Fabrizi M, Johnson R
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(1982): Retrospective assessment of prepubertal major depression with the Kiddie-SADS-E. J Am Acad Child Adol Psychiatry 21:392–397. Peterson SN, Lapetina EG (1994): Platelet activation and inhibition. Novel signal transduction mechanisms. Ann NY Acad Sci 714:53– 63. Pine DS, Wasserman GA, Coplan J, Fried JA, Huagn YY, Kassir S, et al (1996): Platelet serotonin 2A (5-HT2A) receptor characteristics and parenting factors for boys at risk for delinquency: a preliminary report. Am J Psychiatry 153:538 –544. Riess H, Braun G, Brehm G, Hiller E (1986): Critical evaluation of platelet aggregation in whole human blood. Am J Clin Pathol 85:50 –56. Rubin, R (1990): Effects of ethanol on platelets. Lab Invest 63:729 –732. Rubin R, Rand ML (1994): Alcohol and platelet function. Alcohol Clin Exp Res 18:105–110. Schaeffer CF, Brackett DJ, Gunn CG, Dubowski KM (1979): Decreased platelet aggregation following marijuana smoking in man. J Okla State Med Assoc 72:435– 436. Spitzer R., Williams B, Gibbons M. (1987): Instruction Manual
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for the Structured Clinical Interview for DSM-III-R (SCID, 4/1/87 Revision). New York: Biometrics Research Department, NYS Psychiatric Institute. Stewart DG, Brown SA (1995). Withdrawal and dependency symptoms among adolescent alcohol and drug abusers. Addiction 90:627– 635. Tabachnick BG, Fidell LS (1996). Using Multivariate Statistics. Third Edition. New York: Harper Collins College Publishers, p 412. Tarter RE (1990). Evaluation and treatment of adolescent substance abuse: a decision tree method. Am J Drug Alcohol Abuse 16:1– 46. Vanyukov MM, Moss HB, Plail JA, Blackson T, Mezzich AC, Tarter RE (1993): Antisocial symptoms in preadolescent boys and their parents: associations with cortisol. Psychiatric Res 46:9 –17. Wand GS, Waltman C, Martin CS, McCaul ME, Levine, MA, Wolfgang D (1994): Differential expression of guanosine triphosphate binding proteins in men at high and low risk for the future development of alcoholism. J Clin Invest 94:1004 – 1011.
Introduction
P
latelets are activated by a variety of compounds. These include platelet-derived substances such as 5-HT, adenosine diphosphate (ADP), and thromboxane A2 (meFrom the Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (HBM, JKY, KL). Address reprint requests to H.B. Moss, Department of Psychiatry, Temple University School of Medicine, 8th Floor, Jones Hall, 3401 N. Broad Street, Philadelphia, PA 19140. Received November 30, 1998; revised February 1, 1999; revised April 26, 1999; accepted April 27, 1999.
© 1999 Society of Biological Psychiatry
tabolized from arachidonic acid), vasoactive hormones such as epinephrine and vasopressin, connective tissue components such as collagen, peptides such as platelet activating factor, and plasma products such as thrombin and von Willebrand factor. Several well-described steps characterize platelet activation by such agonists: platelet adhesion, dense granular secretion of adenosine triphosphate (ATP), shape change, and finally aggregation (Peterson and Lapetina 1994). Receptors coupling to membrane-associated guanine nucleotide-binding proteins (G-proteins) seem 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 adenylyl 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). We had previously reported preliminary evidence of a diminution in the dense granule secretion of ATP in platelets obtained from a modest sample of abstinent adolescent polysubstance-abusing males with Conduct Disorder in response to treatment with collagen, thrombin, ADP, and ADP plus serotonin (Moss and Yao 1996). We speculated that because this observation of diminished responsivity was independent of the agonist used, the source of such variation might be at the level of cellular signal transduction mechanisms. In that report, effects of comorbid psychopathology were examined and simple bivariate correlations were employed to examine any putative effects of substance use behavior on platelet dense granule secretory responses yielding equivocal results. In this report, we have substantially increased our sample size of adolescents with and without Conduct Disorder who display a wider range of substance use 0006-3223/99/$20.00 PII S0006-3223(99)–112-2
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behavior beyond the history of polysubstance abuse characterizing our original sample. This expanded sample has permitted more informative analyses, and provided the opportunity to directly model the effects of regular use of two important “gateway” drugs, alcohol and marijuana, simultaneously with the platelet effects of Conduct Disorder (CD). These two drugs are of interest as potential mediators of the effect of CD disorder on platelet functioning because substance use behavior and disorders are over-represented among CD adolescents. Alcohol use behavior of adolescents could explain the effect of CD because, to date, at least eight studies have demonstrated reduced agonist-induced platelet aggregation in adult alcoholics entering treatment (reviewed in Rubin and Rand 1994). Similarly, cannabinoids, the active ingredients in marijuana have been shown to have inhibitory effects on agonist-induced platelet aggregation (Formukong et al 1989). As a further extension of the original report, we have also analyzed herein both the aggregation responses of platelets treated with a battery of agonists and their respective dense granule secretory responses. This permits the evaluation of two distinct stages of the aggregation process within a multivariate model framework. Our working hypothesis was that platelets from abstinent CD adolescents would demonstrate diminished platelet dense granule secretory and aggregation responses explicable by alcohol, or marijuana use behavior. Surprisingly, the results suggest an unexpected and complex relationship between CD and marijuana use alone on this platelet model system.
Methods and Materials Subjects Male adolescents with a CD diagnosis (CD⫹; n ⫽ 102) and male adolescents without a CD diagnosis (CD⫺; n ⫽ 101) were recruited by the Center for Education and Drug Abuse Research (CEDAR) from adolescent substance abuse treatment programs, the local juvenile justice system, and from the community. The mean age for the CD⫹ adolescents (⫾SD) was 16.01 ⫾ 0.69 years and the mean age (⫾SD) for the CD-adolescents was 15.49 ⫾ 0.65 years. Community-dwelling subjects were recruited using newspaper advertising, posters, public service announcements, and word-of-mouth. Several of these subjects were part of a longitudinal cohort initially ascertained at age 10 and followed into adolescence. Multiple recruitment approaches were employed in an effort to accrue as representative and broad-based a sample as possible as possible. Exclusionary criteria for all subjects included a past or present history of a psychotic or neurologic disorder, or a full-scale IQ less than 80. The legal guardians of all subjects provided informed consent, whereas adolescents provided informed assent, and were paid for their participation. The Institutional Review Board of University of Pittsburgh Medical Center approved the research protocol.
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Psychiatric Diagnostic Evaluation The adolescents were administered an expanded version of the K-SADS-E (Orvaschel et al 1982) to determine the lifetime presence of DSM-III-R psychiatric disorders including CD. In that the K-SADS does not assess for substance use disorder 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 subject’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 1. The significance of between-group differences in rates of psychiatric diagnoses was determined by Chi-square with continuity correction. In that both alcohol use disorders and cannabis use disorders were also over-represented in CD⫹ youth, more direct measures of the use of these drugs were also examined within the same models.
Measures of Recent Drug Use Two sources of data were used to characterize recent drug use behavior. First, the revised Drug Use Screening Inventory (DUSI-R) 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 10 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 that is then subjected to commercial immunoassay drug screening tests (EZ-Screen, Environmental Diagnostics, Inc.) 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. Subjects who tested positive on the drug screens were not permitted to participate in that day’s research protocol, and were required to return after a week of abstinence. Blood samples were obtained only after a negative drug screen. This effort was designed to eliminate potential confounds of variations in recency of substance use on the biological phenomena of interest.
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 over-arching 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 2 weeks before the scheduled center assessment. No
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Table 1. Common Lifetime Psychiatric Disorders among Conduct Disordered (CD⫹) and Nonconduct Disordered (CD⫺) Adolescents
DSM-III-R disorders:
CD⫹ (n ⫽ 102)
CD⫺ (n ⫽ 101)
Attention deficit hyperactivity disorder
24 (23.5%)
2 (2.0%)
Oppositional defiant disorder
12 (11.8%)
4 (4.0%)
Major depressive disorder
14 (13.7%)
3 (3.0%)
Simple phobias
0 (0%)
2 (2.0%)
Social phobia
2 (1.9%)
0 (0%)
Adjustment disorder
5 (4.9%)
1 (1.0%)
Alcohol use disorder (abuse or dependence)
71 (69.6%)
5 (5.0%)
Cannabis use disorder (abuse or dependence)
65 (63.7%)
4 (4.0%)
Nicotine dependence
14 (13.7%)
3 (3.0%)
8 (7.8%)
2 (2.0%)
12 (11.8%)
1 (1.0%)
Cocaine use disorder (abuse or dependence) Hallucinogen use disorder (abuse or dependence) Inhalant use disorder (abuse or dependence)
8 (7.8%)
enrolled subjects were under treatment with neuroleptics 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 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 blood samples were assayed.
Evaluation of Agonist-induced Platelet Dense Granule Secretion and Aggregation Approximately 450 l of the freshly drawn whole blood was diluted with 450 l of saline in a cuvette containing a disposable siliconized magnetic stirrer. The sample was placed into the Chrono-Log Lumi-Aggregometer (model 500VS) at 37°C and spun at 1000 rpm. To each sample, 100 l of Luciferin (0.16 mg/ml) was added and allowed to equilibrate. Luminescence of a 2-nmol scale as established by adding 5 l 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
0 (0%)
Chi-square (2) (continuity-corrected); df ⫽ 1 2 ⫽ 19.22 p ⬍ .001 2 ⫽ 3.25 p ⫽ .07 (trend) 2 ⫽ 6.31 p ⬍ .01 2 ⫽ 0.52 p ⫽ NS 2 ⫽ 0.48 p ⫽ NS 2 ⫽ 1.52 p ⫽ NS 2 ⫽ 87.84 p ⬍ .001 2 ⫽ 78.15 p ⬍ .001 2 ⫽ 6.31 p ⬍ .05 2 ⫽ 2.58 p ⫽ NS 2 ⫽ 8.11 p ⬍ .005 2 ⫽ 6.31 p ⬍ .05
samples were then stimulated with a variety of weaker agonists, including collagen (2 g/ml), arachidonic acid (0.5 mM), adenosine diphosphate (ADP) (5.0 M), ADP plus a low dose of 5-HT (0.2 g), and ADP plus a high dose of 5-HT (1.0 g). The dose-response of platelet dense granule secretion in response to various agonists has been studied elsewhere (Ingerman-Wojenski 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 before the maximum extent of aggregation (Challen et al 1982). Thus, ATP secretion and aggregation were measured at the time of maximal platelet aggregation for all agonists except thrombin. Platelet aggregation was simultaneously quantified by measuring the change in impedance of the sample against the aggregometer’s internal 20 Ohm standard. Both ATP secretion and impedance changes were measured with an IBM-PC interfaced with AGGRO-LINK (Chrono-Log Corp.) and recorded on a HewlettPackard PaintJet printer.
Statistical Analysis Alcohol and cannabis (marijuana) use information from the DUSI was re-coded into variables indicating either that the subject has “never used” the drug, “uses the drug less than 10 times a month,” and “uses more than 10 times a month.” These responses grouped according to CD status are displayed in Table 2. Although tobacco use behavior was deemed relevant to the
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Table 2. Relationship between Conduct Disorder and Alcohol and Marijuana Use Behavior Self-Reported by Adolescents in the Study
Alcohol use behaviora Never use Regular monthly use Marijuana use behaviorb Never use Regular monthly use
CD⫹ (n ⫽ 102)
CD⫺ (n ⫽ 101)
43 (42.2%) 59 (57.8%)
71 (70.3%) 30 (29.7%)
50 (49%) 52 (51%)
74 (73.3%) 27 (26.7%)
⫽ 16.32, df ⫽ 1, p ⬍ .0001. ⫽ 12.55, df ⫽ 1, p ⬍ .0001.
a 2 b 2
research questions herein, a selective pattern of missing data on this variable precluded its use in these analyses. The measures of alcohol and marijuana use behavior were then dichotomized into a variable indicating either “no use” of the drug or “regular monthly use” to increase factor size and enhance statistical power. A multivariate general linear model approach was employed to simultaneously test the effects of the presence or absence of CD, regular alcohol and regular marijuana use behavior on paired agonist-specific secretory ATP responses and aggregation-associated impedance responses. The distributional characteristics of each agonist-specific secretory ATP response and aggregationassociated impedance change were evaluated for deviation from normality. Logarithmic transformations were employed to reduce skewness for all dense granule secretion and aggregation dependent variables. After the recommendations of Tabachnick and Fidell (1996), we examined the following characteristics for each of the selected models: (a) univariate or multivariate within-cell outlier, (b) normality of dependent variables within cells, (c) homogeneity of variance-covariance matrices within cells, and (d) linearity of the independent-dependent variable relationships within cells. After these adjustments to the variables, their characteristics and relationships were found to be without significant violations of the assumptions noted above. In some cases, there were unequal distributions of participants among variable categories. The distribution of values in cells tended to increase with cell count, that would tend to cause more conservative results (Tabachnick and Fidell 1996). Initially, both main effects and interactions were examined. Non-significant interaction terms were removed from the final models. For significant models, and for thrombin secretory responses alone, univariate F-tests were performed to examine individually the secretory and impedance responses. These analyses were conducted in SPSS for Windows 8.0 (SPSS Inc., Chicago, Ill.) as implemented on a personal computer.
Results Figure 1 demonstrates the relative positions of each of the paired platelet secretory and aggregation responses grouped according to agonist, CD status and marijuana use status essentially summarizing the multivariate analyses.
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As previously noted, only dense granule secretory responses were examined for thrombin, consequently thrombin data are not shown in this graphic representation. As can be seen in the variations in shape and position, different response patterns may be observed for the collagen, arachidonic acid, ADP and the ADP plus serotonin treatments. Collagen and arachidonic acid produce the most robust responses in both the dense granule secretory phase and the aggregation phase. The addition of serotonin shifts the ADP responses to the right along the horizontal axis indicating an augmentation of the aggregation phase, but producing little variation in the dense granule secretion phase. Furthermore, the qualitative effects of CD and marijuana use status differ within each agonist system investigated. Table 3 displays the dense granule secretory and aggregation responses to each of the agonists employed. For thrombin-induced dense granule secretion, a significant interaction of CD status with marijuana use was found (F1,194 ⫽ 4.46, p ⬍ .05), such that platelets from CD⫹ adolescents without regular marijuana use a diminished ATP secretory response compared to CD⫹ regular marijuana users and CD⫺ adolescents. Effects of alcohol use were non-significant. For platelet responses to collagen, a significant interaction of CD status and marijuana use status was found together on the secretory and aggregation responses (F2,194 ⫽ 4.56, p ⬍ 05). For CD⫹ adolescents who were not regular users of marijuana, their platelet secretory responses were less than CD⫹ regular users, but their aggregation responses were greater. For CD⫺ adolescents, regular marijuana users had somewhat greater dense granule secretory response and a greater aggregation response than nonusers of marijuana. Post-hoc univariate F-tests revealed that the interaction of CD by marijuana usage was most pronounced on the aggregation phase of the response to collagen (F1,195 ⫽ 7.67, p ⬍ .01), not the dense granule secretory responses (F1,195 ⫽ 0.84, p ⫽ NS). No effects of alcohol use behavior were noted. For arachidonic acid-induced dense granule secretory and aggregation responses, there was a main effect of regular marijuana usage (F2,169 ⫽ 3.18, p ⬍ .05). Univariate analyses revealed that this effect was predominantly impacting upon platelet aggregation (F1,170 ⫽ 3.42, p ⫽ .06) such that regular marijuana users showed a diminished aggregation response compared to nonusers. No significant univariate effects of marijuana usage upon dense granule secretion were observed (F1,170 ⫽2.17, p ⫽ NS). No effects of alcohol usage were observed. For platelet responses to adenosine diphosphate (ADP), significant interactions of CD status with marijuana use status on the conjoint platelet dense granule and aggregation responses were observed (F2,146 ⫽ 3.62, p ⬍ .05). For
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Figure 1. Relative positions of paired platelet dense granule secretion and aggregation mean values grouped according to Conduct Disorder status (CD⫹ or CD⫺) and regular marijuana use status (MJ⫹ or MJ⫺) for each agonist. The varying shapes of the polygons for collagen, arachidonic acid, ADP and ADP plus 5-HT responses demonstrate the heterogeneity of responses of these biological systems. Collagen and arachidonic acid appear as “strong” agonists, whereas ADP and ADP plus serotonin are “weaker” agonists. The addition of serotonin (5-HT) to ADP shifts responses somewhat to the right primarily along the horizontal axis indicating a predominant effect on aggregation, not dense granule secretion. Significant interactions of CD with marijuana use were found for collagen (p ⬍ .05), ADP (p ⬍ .05), and ADP plus 0.2 g 5-HT (p ⬍ .05). For ADP, and ADP plus 5-HT, the CD⫹/MJ⫺ group shows a lesser agonist response for the dense granule secretory and aggregation phases. For collagen, the CD⫹/MJ⫺ group seems to have a greater aggregation response, but a lessor dense granule secretory response. Main effects of marijuana use were found for arachidonic acid (p ⬍ .005), and ADP plus 1.0 g 5-HT (p ⬍ .05) such that platelets from MJ⫹ subjects have reduced aggregation responses.
CD⫹ adolescents, regular marijuana users had heightened dense granule secretory responses and aggregation responses compared to CD⫹ nonusers of marijuana. For CD⫺ adolescents, marijuana users did not differ from nonusers in terms of dense granule responses, but users had a slightly greater aggregation response than nonusers of marijuana. Post-hoc univariate analyses revealed that interaction between CD and marijuana use status was mostly due to the effects on dense granule secretory responses (F1,147 ⫽ 4.18, p ⬍ .05) and not on aggregation responses (F1,147 ⫽ .00, p ⫽ NS). Effects of alcohol use behavior were not significant. For the platelet responses to ADP plus 0.2 g of serotonin, significant interactions between CD and marijuana use status were also found (F2,156 ⫽ 4.00, p ⬍ .05).
For platelets from CD⫹ adolescents, marijuana users showed an increased dense granule secretory response, but a lessor degree of aggregation than CD⫹ nonusers of marijuana. For platelets from CD⫺ youth, marijuana users had a lesser dense granule secretory response, and reduced aggregation response than nonusers of marijuana. The effect of alcohol use behavior was not significant. For the ADP plus 1.0 g serotonin condition, there was only an effect of marijuana use status observed (F2,154 ⫽ 4.59, p ⬍ .05). Regular marijuana users had somewhat increased dense granule secretion but univariate analysis revealed a significantly reduced aggregation (F1,155 ⫽ 3.70, p ⫽ .06). No effect of alcohol use behavior was observed. Several patterns were also observable in the data in
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Table 3. Mean (⫾SEM) Dense Granule Secretion and Aggregation in Platelets from Adolescent Males with and without Conduct Disorder and Regular Monthly Marijuana Use Conduct disorder (n ⫽ 102) Regular marijuana use (n ⫽ 52)
Agonist Thrombina Collagenb Arachidonic acidc Adenosine diphosphate (ADP)d ADP ⫹ 0.2 g 5-HTe ADP ⫹ 1.0 g 5-HTf
Dense granule secretion (mol ATP)
No conduct disorder (n ⫽ 101)
No marijuana use (n ⫽ 50)
Aggregation (ohms)
Dense granule secretion (mol ATP)
1.92 ⫾ 0.09 1.21 ⫾ 0.06 0.93 ⫾ 0.06 0.51 ⫾ 0.03
NA 29.55 ⫾ 1.33 23.65 ⫾ 1.79 11.45 ⫾ 1.73
0.53 ⫾ 0.06 0.54 ⫾ 0.06
15.58 ⫾ 1.73 18.72 ⫾ 1.92
Regular marijuana use (n ⫽ 27)
No marijuana use (n ⫽ 74)
Aggregation (ohms)
Dense granule secretion (mol ATP)
Aggregation (ohms)
Dense granule secretion (mol ATP)
1.59 ⫾ 0.07 1.06 ⫾ 0.08 0.76 ⫾ 0.06 0.28 ⫾ 0.03
NA 36.66 ⫾ 1.48 27.79 ⫾ 1.97 8.61 ⫾ 1.50
1.96 ⫾ 0.10 1.14 ⫾ 0.07 0.92 ⫾ 0.09 0.54 ⫾ 0.06
NA 30.15 ⫾ 1.71 25.00 ⫾ 1.96 15.58 ⫾ 2.08
1.99 ⫾ 0.07 NA 1.09 ⫾ 0.05 28.93 ⫾ 0.96 0.90 ⫾ 0.05 26.03 ⫾ 1.33 0.54 ⫾ 0.05 14.36 ⫾ 1.38
0.37 ⫾ 0.04 0.39 ⫾ 0.04
19.64 ⫾ 1.96 20.42 ⫾ 1.92
0.54 ⫾ 0.05 0.50 ⫾ 0.06
17.78 ⫾ 2.01 20.26 ⫾ 1.97
0.59 ⫾ 0.05 20.05 ⫾ 1.15 0.53 ⫾ 0.04 22.34 ⫾ 1.20
Aggregation (ohms)
Significant multivariate main effects and interactions: a CD ⫻ Marijuana: F 1,194 ⫽ 4.46, p ⬍ .05. b CD ⫻ Marijuana: F 2,194 ⫽ 4.56, p ⬍ .05. c Marijuana effect: F 2,169 ⫽ 3.18, p ⬍ .05. d CD ⫻ Marijuana: F 2,146 ⫽ 3.62, p ⬍ .05. e CD ⫻ Marijuana: F 2,156 ⫽ 4.00, p ⬍ .05. f Marijuana effect: F 2,154 ⫽ 4.59, p ⬍ .05.
Table 3. Generally, platelet dense granule secretion and aggregation responses across agonists tended to be lowest in the CD⫹ no regular marijuana use group. Platelet dense granule responses in the CD⫹ regular marijuana use group resembled those of the CD⫺ no marijuana use group.
Discussion This study extends, but does not replicate our original observation of a variation in platelet dense granule secretion in CD⫹ adolescent males with polysubstance abuse. In this enriched sample, we have employed multivariate techniques to model the simultaneous effects of CD, alcohol use behavior and marijuana use behavior on both phases of the platelet responses to several established agonists. These results suggest a much greater complexity in the determinations of both phases of platelet responses than assumed in the original report of polysubstance abusing CD⫹ males. Herein, the presence or absence of CD was found to significantly interact with marijuana use behavior in a manner that was fairly independent of the specific agonist system examined. Significant interactions between CD status and marijuana use status were found for responses to thrombin, collagen, ADP, and ADP plus 0.2 g of 5-HT. Thus, the impact of regular marijuana usage on platelet responsivity to these agonists depended upon whether the subject was either CD⫹ or CD⫺. To add further complexity to the problem, many of the effects of this interaction were observed to be more pronounced in either the dense granule secretory phase or the aggregation
phase of the response again depending upon the agonist system. The least pronounced platelet responses in both phases were found in samples from adolescents with CD, but who did not use marijuana. Curiously, with the exception of collagen responses, platelet responses from marijuana using, CD⫹ adolescents tended to resemble those from CD⫺, non-marijuana using subjects. The results suggest that platelets from CD⫹ youth, that do not regularly use marijuana, demonstrate a variation in responsivity, regardless of the identity of the agonist employed. Because platelet receptor coupling to membrane-associated guanine nucleotide-binding proteins (Gproteins) is a common feature of platelet surface receptors (Peterson and Lapetina 1994), differences in plasma membrane or G-protein function could account for the observed variations in platelet responses. In a recent 31P magnetic resonance spectroscopy study, we reported a regional reduction of phosphodiesters in the brains of prepubertal children with CD or Oppositional Disorder (Moss et al 1997). This reduction in the breakdown products of membrane phospholipids could reflect a more global variation in the composition of plasma membranes. On the other hand, Wand et al (1994) suggested that variations in signal transduction mediated through the differential expression of the stimulatory G-protein might provide a biological substrate for the familial liability to alcoholism. Cellular activation reactions common to most platelet agonist receptors include second messenger effects such as activation of adenylyl cyclase, the stimulation of phospholipase activity, calcium mobilization, the phosphoinositide cascade reaction, phosphorylation of regulatory proteins
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by kinases, the formation of new messengers and discharge of storage granules (Colman 1990; Rubin 1990). Biological variations due to CD could be localized to any of these intra-cellular processes. Investigations of these putative mechanisms await further research. Pine et al (1996) have reported platelet serotonergic variations such as reductions in the density of the platelet 5-HT2a receptor in delinquent adolescents. The magnitude of serotonin augmentation of ADP-induced platelet aggregation, as employed herein, has been demonstrated to reflect the density of platelet 5-HT2a receptors (McBride et al 1987). Consequently, if one examines only the 5-HT augmentation of the ADP platelet response, the results might imply a specific reduction in 5-HT2a receptors among adolescents with CD. The observation of this effect across multiple agonists lends less credence to the solitary interpretation. Parsimony suggests that the variations in platelet responses among CD⫹/MJ⫺ youth across multiple agonists are less likely to be based on individual effects localized to specific receptors. It is more probable that these multiple agonist effects are due to factors common to these varied receptors, either at the level of the biological membrane or intracellular signaling mechanisms. It is tempting to speculate that the biological substrate mediating the under-responsivity of platelets might also mediate other aspects of under-reactivity that has been hypothesized to be the basis for the development of deviant behavior, including conduct disorder and antisocial personality disorder. Eysenck (1977) has argued that adult antisocial criminals have an under-aroused 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 neuroendocrine response to pharmacological challenge with a serotonin agonist (Moss et al 1990). In addition, we have observed in children a hyporesponsivity of the hypothalamic-pituitary-adrenal axis to anticipated stress that was associated with higher conduct disorder symptom counts and paternal antisociality (Vanyukov et al 1993; Moss et al 1995; Moss et al 1999). Such under-reactivity may be either constitutive (reflect a higher genetic predisposition), or result from an adaptation to prolonged stress, perhaps caused by parental antisociality (e.g., high aggression) and substance abuse, or an interaction of the two. Pharmacological effects of marijuana on platelet responsivity are less obscure and better supported by clinical and pre-clinical investigation. Schaeffer et al (1979) reported a decrease in platelet aggregation among marijuana smokers. Formukong et al (1989) subsequently examined
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the effects of adding several cannabinoid constituents of marijuana to human and rabbit platelets and observing their activation in vitro. Olvetol, cannabidiol, cannabinol and tetrahydrocannabinol were all found to inhibit adrenaline-induced platelet aggregation in a dose-dependent fashion. Only olvetol inhibited phorbol ester-induced aggregation limiting the possibility that all cannabinoid effects are at the level of protein kinase C. The observation that cannabinoids were effective inhibitors of platelet activating factor-induced aggregation suggests that these effects may be on the prostaglandin cascade, perhaps through inhibition of cyclo-oxygenase. The method used by these investigators was different from that employed by us in this report. Formukong et al. used an aggregometer that was based on changes in light transmission produced by the clumping of platelets. Their results may be interpreted as being analogous to our impedance aggregation measure, but not our dense granule secretion measure. More recently, Skosnik, Yao and Park (unpublished data), using dense granule secretion and impedance aggregation measures, have shown that the addition of two endogenous cannabinoids to blood produced an inhibition of ADPinduced (but not collagen or arachidonic acid) platelet aggregation. Furthermore, they have noted a hyper-responsivity of platelets from schizophrenic patients compared to controls in terms of cannabinoid inhibition of aggregation demonstrating an interaction between psychopathology and the cannabis effects on this peripheral model system. Our data are not totally consistent with the in vitro studies, although some inhibition seems to be appreciable in terms of the second phase of aggregation for arachidonic acid responses and the ADP plus 1.0 g serotonin responses. The discrepancy between the in vitro studies this clinical investigation may be due to the fact that our subjects were required to be abstinent for 72 hours before blood sampling. The life-span of the human platelet is 7–10 days (Nathan 1988), thereby providing a window of opportunity for physiologic alteration by highly lipophilic cannabinoids. Thus, any effects noted herein may be attributable to chronic exposure to marijuana rather than acute effects of cannabinoids. It was admittedly surprising that we were not able to find an effect of alcohol use on platelet aggregation. There is a significant extant literature reporting the impact of alcoholism on platelet functioning in adults (Rubin and Rand 1994). Nonetheless, we found no effects of alcohol use behavior on any of the agonist systems studied, perhaps due to the differences in the alcohol use behaviors and clinical presentations of adolescent alcohol abuser compared to adult alcoholics. Adolescents typically binge drink, whereas daily intoxication is more frequently found among adult alcoholics. (Stewart and Brown 1995; Martin et al 1995). Thus, the short life span of the platelet,
Platelet Responses in Conduct Disorder
combined with more sporadic alcohol exposure, and the 72-hour abstinence requirement might have limited our ability to detect alcohol effects on platelet physiology. There are several important limitations in this study that impacts upon its interpretation and generalizability. First and foremost is the selectivity of the adolescent sample. The majority of CD cases were obtained through adolescent substance abuse treatment programs, and the local juvenile justice system. Thus, they may represent a more extreme variant due to Berksonian bias (Berkson 1946), and consequently have a greater probability of a constitutive difference from community-dwelling subjects. Second, our sample size is not sufficiently large to adequately model and test any platelet effects due to comorbid psychiatric disorders. As can be seen in Table 1, there is substantial psychiatric co-morbidity in the sample. Larger cell sizes for each comorbid combination would be necessary to permit informative multivariate analyses. In our previous report, we used univariate methods to rule out effects of attention deficit disorder, oppositional defiant disorder, and major depression (Moss and Yao 1996). Third, self-report information on drug use behavior, especially among conduct disturbed adolescents maybe be inaccurate due to response biases in this population. Fourth, our diagnoses are based upon DSM-III-R, as opposed to DSM-IV. Thus, we cannot accurately subtype our CD adolescents into childhood versus adolescent onset Conduct Disorder in accordance with DSM-IV criteria. Lastly, inconsistencies in the collection of data on cigarette smoking behavior (as opposed to the presence or absence of nicotine dependence) precluded an adequate examination of the effects of tobacco smoking on platelet aggregation responses. Because nicotine dependence is over-represented in the CD⫹ group, we cannot rule out effects of nicotine, tars, or carbon monoxide on platelet functioning. We hope to examine effects of smoking more directly employing a newly accrued sample of adolescents. Similarly, hallucinogen use disorders are over-represented in the CD⫹ group, but the cell size is not sufficiently large to permit meaningful analyses. This work was performed under the auspices of the Center for Education and Drug Abuse Research (a consortium of the University of Pittsburgh and St. Francis Medical Center) (P-50-AA 08746 and supported by the National Institute on Drug Abuse (P50-DA 05605; Dr. Moss DA-00308). Dr. Lynch is supported by National Institute on Alcohol Abuse and Alcoholism (P-50-AA 08746).
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