The Procoagulatory Effects of Delta-9-Tetrahydrocannabinol in Human PlateletsEngelbert Deusch, MD, Hans Georg Kress, MD PhD, Birgit Kraft, MD and Sibylle A. Kozek-Langenecker, MD
+ Author Affiliations
Department of General Anesthesiology and Intensive Care B, Vienna Medical University, Vienna, Austria
Address correspondence to Engelbert Deusch, MD, Department of General Anesthesiology and Intensive Care B, Vienna Medical University, General Hospital Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria, E.C. Address e-mail to firstname.lastname@example.org.
A & A October 2004 vol. 99 no. 4 1127-1130
Delta-9-tetrahydrocannabinol (THC) is increasingly used for the long-term treatment of nausea, vomiting, cachexia, and chronic pain. Recent reports, however, have indicated an increased risk of myocardial infarction and thromboangiitis obliterans after THC intake. Blood platelets have an essential role in the pathogenesis of these two diseases, but it is unclear whether platelets are potential target cells for cannabinoids. We investigated the effects of THC on human platelets and the expression of cannabinoid receptors on their cell membranes in this in vitro study. The effects of THC (final concentrations 10−7 to 10−5 M) on the expression of activated platelet fibrinogen receptor (glycoprotein IIb-IIIa) and P selectin were characterized by flow cytometry. Western blotting was performed with platelet membrane preparations to determine the surface expression of cannabinoid receptors on human platelets. THC increased the expression of glycoprotein IIb-IIIa and P selectin on human platelets in a concentration-dependent manner. The two known cannabinoid receptors (CB1 and CB2) were both detected on the cell membrane of human platelets. Our functional results may suggest a receptor-dependent pathway of THC-induced platelet activation. However, further in vivo studies are warranted to evaluate the role of cannabinoid receptors in mediating the demonstrated procoagulatory effect of THC.
IMPLICATIONS: Delta-9-tetrahydrocannabinol activated human platelets in vitro in a concentration-dependent manner. The two known cannabinoid receptors (CB1 and CB2) were both detected on the cell membrane of human platelets.
Delta-9-tetrahydrocannabinol (THC) is a major compound of natural cannabis, which is increasingly used for the palliative treatment of patients with malignancies or chronic pain because of its strong antiemetic and appetite-stimulating effects in conjunction with its analgesic effects (1). Several reports, however, indicate an association of chronic THC intake and myocardial infarction (2) as well as the juvenile onset of thromboangiitis obliterans (3). Blood platelets have an essential role in the pathogenesis of the two diseases (4,5), but it remains unclear whether platelets are involved in the pathogenesis of THC-triggered prothrombotic adverse events. Moreover, it is poorly understood whether platelets are target cells for cannabinoids. Although it is well accepted that platelets synthesize endogenous cannabinoids (6), previous studies on the effects of THC on platelet function revealed contradictory results. Some authors reported an inhibiting effect of large concentrations of THC (≥10−5 M) on agonist-induced platelet aggregation (7); others found increased spontaneous aggregate formation in the presence of THC (8). To clarify this issue, we investigated (a) the existence of cannabinoid receptors on the platelet surface membrane by means of Western blotting, and (b) the effect of THC on platelet function at the cellular level using whole blood flow cytometry for the detection of platelet surface activation markers (9).
After IRB approval and informed consent, venous blood specimens from eight healthy volunteers were studied. All participants denied the use of any drugs within the preceding 14 days. Blood was drawn into Vacuette™ tubes (Greiner, KremsmÃ¼nster, Austria) containing 3.8% trisodium citrate (9:1 v/v) from the antecubital vein by venipuncture without stasis. The first 3 mL was always discarded. Citrate was used as an anticoagulant because of its negligible intrinsic effects on platelets (10). All samples were processed within 3 min using 5-mL polystyrene round-bottom tubes (Falcon™; Becton Dickinson, Franklin Lakes, NJ).
Blood samples were diluted (1:5) in Dulbecco’s phosphate-buffered saline free of Ca2+, Mg2+, and NaHCO3 (Na2HPO4 1.15 g/L, NaCl 8 g/L, KH2PO4 0.2 g/L, KCl 0.2 g/L, pH 7.3; Life Technologies, Paisley, UK) to inhibit the contact between individual platelets. Aliquots of diluted blood were incubated without and with THC at 10−7, 10−6, and 10−5 M final concentrations. These concentrations are within the clinical concentrations measured after oral ingestion of 20 mg of THC (unpublished data) and less than plasma peak concentrations measured after THC smoking (11).
Platelet activation transforms platelet membrane glycoprotein IIb-IIIa complexes into a conformational state that is competent for binding fibrinogen (12). P selectin, which is expressed on the surface of activated platelets as the internal α-granule membrane becomes integrated into the cytoplasmic membrane (13), mediates heterotypic aggregate formation, and serves as a marker for platelet secretion and activation. Expression of activated glycoprotein IIb-IIIa and P selectin was assessed with the monoclonal antibodies PAC-1, anti-CD62, and anti-CD61(Becton Dickinson Immunocytometry Systems, San Jose, CA), and flow cytometry was performed using a stain-and-fix technique (14) on 25.000 gated events. In each experiment, one sample was stained with isotype-matched, nonspecific mouse immunoglobulins to set a threshold for fluorescein isothiocyanate- and phycoerythrin-positive cells. Platelets were selectively gated by logarithmic forward versus side light scatter plots using the CD61 platelet-specific antibody. Histograms of FL-1 or FL-2 were generated, and both the mean fluorescence intensity of PAC-1 (arbitrary units) and the percentage of anti-CD62P-positive platelets were determined with CellQuestPro™ software on a FACSCalibur™ flow cytometer (Becton Dickinson). Flow cytometric analysis was performed in duplicate samples.
Western blotting was performed on platelet membrane preparations to identify surface cannabinoid receptors. Platelet membrane preparations were prepared from a leukocyte-depleted platelet concentrate as described elsewhere (15). The protein concentration was assessed by Biorad Protein Assay (Bio-Rad™, MÃ¼nchen, Germany). Proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel. Proteins were transferred on a nitrocellulose membrane (pore size 0.45 μm, Protean™ BA 85; Schleicher & Schuell, Dassel, Germany). The two cannabinoid receptors CB1 and CB2 were identified with affinity pure polyclonal rabbit anti-human cannabinoid receptor 1 or 2 antibodies (Alpha Diagnostics Intl., San Antonio, TX). A goat anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (1:2500; Amersham Bioscience, Uppsala, Sweden) was detected by chemoluminescence (Supersignal™; Pierce, Rockford, IL).
The significance of the concentration-dependent effect of THC was assessed by one-way analysis of variance. P < 0.05 was considered statistically significant. Data were expressed as arithmetic means Â± SD.
The expression of activated glycoprotein IIb-IIIa (P < 0.05 at ≥10−6 M THC) and P selectin (P < 0.05 at ≥10−7 M THC) on human platelets was significantly enhanced by THC in a concentration-dependent manner when compared with control values (Fig. 1A). Platelet membrane preparations were positive for both CB1 and CB2 (Fig. 1B). Control experiments with the secondary anti-immunoglobulin G antibody alone were negative (Fig. 1B, control panel).
Figure 1. A, Effect of delta-9-tetrahydrocannabinol (THC) on the expression of activated platelet glycoprotein IIb-IIIa and P selectin as evaluated by using the monoclonal antibodies PAC-1 and anti-CD62, respectively. THC increased the expression of glycoprotein IIb-IIIa and P selectin on platelets in a concentration-dependent manner. Independent experiments (n = 8) were performed in duplicate determinations. Mean Â± SD. *P < 0.05 compared with control samples without THC. B, Determination of the existence of cannabinoid receptors on platelet membrane preparations by means of Western blotting. Platelet membrane preparations were positive for both cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 was predominantly expressed in human platelet membranes. Antibodies against CB1 also seem to detect CB2 to a small extent, as the upper band corresponds to the molecular weight of CB2. Control experiments with the secondary anti-immunoglobulin G antibody alone were negative.
In the current study, the effect of THC on platelet reactivity was determined by assessing platelet membrane receptor expression as an index of platelet function. Results presented in Figure 1A clearly demonstrate that THC activates platelets as determined by enhanced glycoprotein IIb-IIIa expression and P selectin expression. The present results demonstrate that platelets are potential target cells for cannabinoids. Moreover, Western blotting revealed CB1 and CB2 on the platelet surface (Fig. 1B), which have not yet been investigated. Together, these results may suggest a receptor-dependent pathway of THC-induced platelet activation. However, further experiments are warranted to evaluate the role of cannabinoid receptors in mediating in the demonstrated procoagulatory effects of THC.
Endogenous ligands of cannabinoid receptors are endocannabinoids like anandamide and 2-arachidonoyl-glycerol (16). Comparable to the observed platelet-activating effect of the exogenous natural cannabis compound THC, anandamide induced aggregation of washed human platelets (at concentrations of 10−5 M) (17). Another endogenous cannabinoid, 2-arachidonoyl-glycerol, also binds and activates human platelets (at concentrations of 2 Ã— 10−4 M) (18). These findings, together with the present proof of the existence of cannabinoid receptors on the platelet membrane and the detection of genuine platelet-activating effects of THC in the physiological test milieu of whole blood using flow cytometry, confirm the hypothesis that platelets are target cells for cannabinoids. The physiological role of this interaction remains to be determined.
Extrapolation from in vitro experiments to clinical practice must be made with care. Nevertheless, the clinical consequence of increased platelet adhesion molecule expression may be that THC administered in vivo triggers coagulation problems. Indeed, several case reports indicate the procoagulatory adverse effects of cannabinoids (2,3): THC intake by smoking marijuana was found to be associated with juvenile onset of thromboangiitis obliterans (3), and to increase the risk of myocardial infarction (2), which was potentiated fivefold in the first hour after smoking marijuana (2). Even though THC may increase morbidity, two epidemiological studies in young populations showed no or only little increased mortality among marijuana users (19,20). Unfortunately, these studies give no hint on the role of the preexisting health status of marijuana users in the risk assessment for procoagulatory adverse events. It may be speculated, though, that the risk is even higher during long-term cannabis use and in seriously ill patients with compromised cardiovascular reserve. Especially in this population, the use of cannabis for medical purposes is currently the focus of clinical and scientific interest. Although clinical relevance remains controversial (21), THC is increasingly used for the palliative treatment of patients with malignancies or chronic pain because of its strong antiemetic and appetite-stimulating effects in conjunction with its analgesic effects (1).
Aside from the observed activation of primary hemostasis, adverse side effects of THC on the cardiovascular system may contribute to the pathogenesis of thromboangiitis obliterans and myocardial infarction. Among them, a concentration-dependent increase in heart rate and arterial blood pressure, leading to an increased myocardial workload and oxygen demand, have been documented (22). Compromised oxygen supply by the increased generation of carboxyhemoglobin during marijuana smoking further deteriorates the myocardial oxygen balance (23). Clinical in vivo studies are warranted to determine the relevance of these potential cardiovascular and coagulation side effects of THC in seriously ill patients. Until then, therapists must be aware of the potential adverse effects of THC.
This work was supported by Ã–sterreichische Nationalbank JubilÃ¤umsfonds Grant 9583.
S. Sonneborn, Humanplasma GmbH, Vienna, Austria donated platelet concentrates.
Accepted April 21, 2004.
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