The effect of THC pretreatment on LPS-stimulated TF procoagulant activity of monocytes was measured over the course of 24 h. Peak TF activity occurred after 6 h of LPS stimulation independent of THC treatment (Fig. 1a). By 24 h, TF procoagulant activity remained elevated in THC-treated LPS-stimulated monocytes, but declined significantly in vehicle-treated or untreated LPS-stimulated cells (Fig. 1a). Similar to monocyte TF activity, peak microvesicle TF activity occurred by 6 h of LPS stimulation (Fig. 1b). After 24 h, TF activity of microvesicles from THC-treated cells was approximately 3-fold greater compared to vehicle-treated or untreated LPS stimulated MVs (Fig. 1b). Although the magnitude of monocyte and microvesicle TF activity was donor dependent, THC-mediated increased TF activity occurred with a range of 2- to 6-fold at 24 h among donors. Results indicate that THC modulation of TF activity in monocytes was paralleled by elevation in microvesicle TF activity.

To confirm that TF activity was the result of elevated TF protein expression and not dependent on a co-factor or post-translational modification, TF protein levels were evaluated by western blot. TF protein appeared by 2 h post LPS stimulation (Fig. 1c). No differences in levels were apparent until 24 h of LPS stimulation, when THC-treated cells had greater levels of TF protein compared to cells treated with vehicle control. As expected, TF protein was undetectable in the absence of LPS stimulation (data not shown). Next, TF mRNA levels were evaluated over 24 h by qPCR. TF mRNA levels in THC-treated and vehicle control-treated cells remained similar until peak expression 2 h post LPS stimulation, but were elevated in THC-treated cells at all subsequent time points (Fig. 1d). Among 5 donors, levels of TF mRNA in the presence of THC were approximately 2-fold higher at 3 and 6 h, increasing to 4-fold higher by 12 and 24 h (Fig. 1e). Although the magnitude of THC responses differed among donors, all donors showed THC-mediated elevated TF expression from 2 to 5-fold relative to control over 3 to 24 h of LPS stimulation (Fig. 1e). Findings indicate that THC treatment prolongs levels of TF mRNA and protein, as well as TF procoagulant activity, in both monocytes and monocyte-derived microvesicles.

Enhancement of monocyte TF protein levels by THC was dose dependent with maximal production at the 30 μM dose (Fig. 2a, b). Monocyte TF activity increased 250 % at the 30 μM dose (Fig. 2c). Similar to monocyte TF activity, LPS-stimulated microvesicle TF activity showed a dose dependent THC mediated elevation (Fig. 2d). Similar numbers of microvesicles were observed by flow cytometry between THC- and vehicle-treated or untreated LPS-stimulated supernatants (Fig. 2e), indicating that TF activity increased per microvesicle rather than via increased microvesicle release by THC-treated LPS-stimulated cells. Concentrations of THC used were higher than levels reported in the circulation of individuals who use marijuana, but similar to other ex vivo studies of THC [17–20], perhaps reflecting serum reduction of bioactivity by THC in tissue culture models [21].

To explore the mechanism by which THC enhanced LPS-stimulated TF expression and activity, selected signal transduction events were investigated. Although TF expression is dependent on ERK1/2 and NF-κB signaling [2, 3], phosphorylation of ERK1/2 and p65 or degradation of IκBα were unchanged (Fig. 3a-d). Since signal transduction is unaltered, it is unlikely that THC ubiquitously promotes inflammation, rather THC imparts an effect that is TF specific. Moreover, TNFα or IL-8 mRNA and secreted protein increased modestly with THC treatment, while IL-6 was unchanged (Fig. 4). In addition, while THC treatment results in modest increases in IL-8 and TNFα, THC mediated elevations in TF are of a greater magnitude. However, as TNFα stimulates TF expression [22], small elevations in TNFα expression may act synergistically with other mechanisms to further enhance THC mediated elevations in TF expression and activity.

Our results are consistent with a mechanism of THC mediated elevation of TF expression at a post-transcriptional level by inducing stabilization or preventing degradation of TF mRNA. Recently, Poly(ADP-ribose)-polymerase(PARP)-14 and tristetraprolin (TTP) were shown to cooperate to mediate TF mRNA degradation [5]. While TTP regulates mRNA transcripts of inflammatory mediators, such as TNFα [23], the addition of PARP-14 renders the complex TF specific. Since the magnitude and kinetics of TF mRNA expression (Fig. 1e) differ from TNFα (Fig. 4b), our data suggest that TTP likely plays less of a role in THC mediated elevations of TF compared to PARP-14.

Recreational marijuana use is prevalent, including among individuals with IBD and HIV-1 infection [24, 25], who are also at increased risk for coagulation disorders [8, 10]. In addition, marijuana users are likely to consume alcohol [25]. Both acute binge drinking and chronic alcohol use increase microbial translocation and circulating endotoxin [26, 27]. Taken together with our results, marijuana use alone or coupled with excessive alcohol use, may also enhance circulating procoagulant capacity.

Findings indicate that marijuana use may increase the procoagulant potential of circulating monocytes and underscore the importance of investigating the effects of marijuana use in vivo. Recently, several cases of sudden death in otherwise healthy individuals have linked acute marijuana use to cardiovascular complications [28]. As use of marijuana for both medicinal purposes and recreational purposes increases, investigation and close monitoring of coagulation related disorders is crucial, especially in individuals with diseases characterized by microbial translocation and dysregulated systemic inflammation.