Increased platelet activation in HIV patients is not altered with reported cocaine use.

<p>Blood samples were collected from HIV-negative, healthy subjects (HIV-, N = 61), HIV-positive individuals without cocaine use (HIV+Coc-, N = 37), and HIV-positive individuals with reported cocaine use within one year (HIV+Coc+, N = 16), and were fixed and stained as described in the methods section. Platelet CD62P expression was significantly higher in HIV+Coc- and HIV+Coc+ samples as compared to HIV- subjects (* p = 0.038, ** p = 0.008, respectively). CD62P expression levels between HIV+Coc- and HIV+Coc+ samples were statistically similar (ns, p = 0.492). Data are represented as fold change in CD62P MFI as compared to HIV- subjects and are shown as mean ± SEM.</p

Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse

<div><p>Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV) infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB) inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK) activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and <i>in vitro</i> kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated <i>ex vivo</i>. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders.</p></div

Cocaine sensitizes platelets from HIV+, but not HIV-, donors to activation by ADP.

<p>Whole blood from healthy subjects (HIV-, N = 4) or HIV-positive subjects (HIV+, N = 4) was either left untreated (NT) or was treated with the indicated concentrations of cocaine alone or together with the indicated concentrations of ADP for 30 minutes. Platelet activation in whole blood was assessed via CD62P staining. In the HIV+ samples there was a significant increase in platelet activation following treatment with 10 μM ADP plus cocaine (*** p<0.001 compared to 10 μM ADP alone in the HIV+ samples, ^### p<0.001 compared to 10 μM ADP plus cocaine in the HIV- samples). Treatment with 1 μL DMSO for 5 minutes followed by 1 μL dH₂O for 30 minutes was used as a vehicle (Veh) control. Data are represented as fold change in CD62P MFI as compared to NT samples and are shown as mean ± SEM.</p

HIV-induced platelet activation does not involve NF-κB signaling mechanism.

<p>(A) Platelet lysates collected from HIV-negative subjects (HIV-, N = 2) and HIV-positive subjects (HIV+, N = 2) were subjected to electrophoretic mobility shift assays followed by supershift with anti-p50 and anti-RelA antibodies. Incubation with pre-immune serum was included as a control. Both p50 and RelA antibodies altered the mobility of bands, suggesting the presence of these two molecules in NF-κB/DNA complexes, however, no apparent differences were found between HIV- and HIV+ samples. (B) Platelet lysates collected from HIV-negative, healthy subjects (HIV-, N = 11), HIV-positive patients without cocaine use (HIV+Cocaine-, N = 10), and HIV-positive patients with reported cocaine use within one year (HIV+Cocaine+, N = 8) were subjected to immunoblot analysis with antibodies against IKKα, IKKβ, IκBα, or α-Tubulin. Expression levels of IKKα and IKKβ were noted to be similar between the groups. There was an increase in IκBα phosphorylation as evidenced by the appearance of a less mobile, upper band (*), which was confirmed by densitometric analysis of this upper phospho-IκBα band as shown in (C). (D) Platelet lysates collected from HIV- subjects (N = 15), HIV+Coc- (N = 10), and HIV+Coc+ (N = 5) were subjected to immunoprecipitation with an anti-IKKβ antibody followed by an IKKβ <i>in vitro</i> kinase assay. Incorporation of [³²P] into the recombinant IκBα substrate was measured by densitometric analysis of autoradiograms. No significant differences in IKKβ activity between the sample groups were noted. Data are represented as fold change in IKKβ activity compared to the HIV- samples and are shown as mean ± SEM.</p

Cocaine indirectly activates platelets <i>in vivo</i>.

<p>Wild-type C57BL/6 mice were injected intraperitoneally (IP) with either PBS (N = 12), 5mg/kg cocaine (N = 6), or 50mg/kg cocaine (N = 6) three times per week for three weeks. Following the final injection, blood was collected into tubes containing acid citrate dextrose, and platelet activation in whole blood was assessed via CD62P staining. Treatment with both concentrations of cocaine resulted in increased platelet activation as compared to the PBS treated mice (* p<0.05, ** p<0.01). Data are represented as fold change in CD62P MFI as compared to PBS treated samples and are shown as mean ± SEM.</p

Cocaine and HIV do not activate platelets in whole blood when treated <i>ex vivo</i>.

<p>Whole blood from healthy subjects (3 to 4 donors) was left untreated (NT) or was treated with cocaine (A), infectious NL4-3 HIV (B), ADP or Tat (C) (D), for 30, 60, and 30 minutes respectively, or as indicated, following which CD62P expression was assessed as a marker of platelet activation via flow cytometry. Treatment with cocaine, HIV, and Tat did not result in increased platelet activation. In each experiment, as indicated, treatment with 1 μL dH₂O for 30 minutes was used as a vehicle (Veh) control. Data are represented as fold change in CD62P MFI as compared to NT samples and are shown as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Demographic and clinical characteristics of study participants.

<p>HIV-negative, healthy controls (HIV-, N = 61), HIV-positive individuals without cocaine use (HIV+Coc-, N = 37), and HIV-positive individuals with reported cocaine use within the previous one year (HIV+Coc+, N = 16) were enrolled in the study. All HIV-positive individuals were on antiretroviral therapy at the time of the blood draw. Unless otherwise stated, <i>N</i> indicates sample number with percentage of total sample group in parentheses. NA indicates not applicable.</p><p>Demographic and clinical characteristics of study participants.</p

Substance Use Characteristics.

<p>Substance Use Characteristics.</p

Principal component analysis of features identified by machine learning pipeline.

<p>Principal component analysis (PCA) of 19 HCs (black circles) and 19 IDUs (red asterisks) based on ten features from the various assays identified by machine learning (CD19+CD20low/negIgD-CD27-, MIP-1β, gp140 IgM, IgG4, CD19+CD20+, sCD40L, TGF-α, MH: d18:1/16:0, TNF-α and MH: d18:1/22:0). PCA was computed on per-feature z-scores (based on all 38 samples) of log transformed data (except for B cell subset frequencies which were not log-transformed).</p

Plasma analyte profile.

<p>Plasma cytokines, chemokines and growth factors were measured by Milliplex Immunoassay and LPS was measured by a limulus assay. (A) Analyte profiles are shown as a heat map similar to above. Analyte concentrations were log-transformed prior to computing z-scores. (B) Select plots of individual analytes, each symbol represents an individual subject, the red lines indicate mean.</p