1
Supplementary Material and Methods
Patients and healthy volunteers
Samples from patients and healthy controls were obtained under NIAID Institutional
Review Board approved clinical research study protocols in the NIAID/CCMD
intramural program. Both healthy controls and HIV infected patients supplied written
informed consent for use of their blood. Samples from healthy controls were obtained
from the NIH Blood Bank. Samples from patients with HIV infection were obtained
from the NIH Clinical Center. Patients and healthy controls used in Figure 1, 2, 3 and 4
are described at Table S1. Treatment regime and associated complications of patients on
Figure 2, 3, 4, are presented in Table S3 and S4.
Flow cytometry
Whole blood was collected and processed within 3 hours after blood draw. For whole
blood staining, 400 µl blood was incubated for 10 minutes at room temperature with 10
µg/ml human IgG (Sigma-Aldrich, MO) to prevent potential Fc receptor binding. This
was followed by incubation with a cocktail of mAbs: anti-CD3 FITC (BD Pharmingen,
clone SK7); anti-CD4 Qdot605 (Invitrogen, clone S3.5) or anti-CD8 Qdot605
(Invitrogen, clone 3B5); anti-CD45RA Pacific Blue (Invitrogen, clone MEM-56); anti-
CD27 APC-H7 (BD Pharmingen, clone M-T271); anti-CD42b PerCP (Biolegend, clone
HIP1); anti-CD62P P-Selectin APC (BD Pharmingen, clone AK-4). After a 15 minute
incubation at room temperature, red blood cells were lysed with BD FACS Lysing Buffer
(BD Biosciences, CA). Samples were collected on a BD LSR II using FACSDiva
software and the data was subsequently analyzed using FlowJo software (Tree Star).
Binding Recombinant CD62P-Fc: T cells were obtained from PBMCs from healthy
controls (n=13) and HIV infected patients (n=18). cART treated HIV infected patients
2
had successfully suppressed viremia to <40 copies/ml (median months cART 65 (IQR:
44.8-135.3) and supplementary Table S1). T cells were isolated by negative selection
(Pan T cell isolation kit, Miltenyi Biotec, CA). Purified T cells were rested overnight in
X-Vivo Medium (Lonza, MD). Cells were washed twice with binding buffer (HBSS +
Ca++ Mg++ + 1 mg/ml BSA) and incubated for 30 minutes at 40C with 10 µg/106 cells of
recombinant human P-selectin/CD62P (Trp42-Ala771)-Fc chimera (CD62P-Fc) or
recombinant human IgG Fc as control (R&D System, CA). CD62P-Fc binding was
performed in absence or presence of 10 mM EDTA. After incubation, cells were washed
twice and detection was done using a mouse anti-human IgG Fc APC labeled (Southern
Biotech, clone H2). After washing, the cells were stained with surface markers for T cell
subsets as above.
Thrombin stimulations: PBMCs were isolated by ficoll gradient centrifugation. PBMCs
were stimulated with 50U/ml thrombin and analyze by flow cytometry or Amnis (EMD-
Millipore, WA) as described in Supplementary material and methods.
Biomarkers
Serum levels of D-dimer and soluble CD62P were assessed in HIV infected patients
(n=20) and healthy controls (n=23). The D-dimers were run using an ELFA (Enzyme-
Linked Fluorescence Assay) on a VIDAS instrument (BioMerieux, Durham, North
Carolina). Soluble CD62P serum levels were measured with a magnetic bead Milliplex
on a Luminex xMAP (EMD Millipore, CA).
Thrombin stimulation: 3x106 total PBMCs were resuspended in stimulation buffer
(HBSS + Ca++ Mg++ + 1 mg/ml BSA) and stimulated for 5 or 15 minutes at 370C with 50
U/ml of thrombin (Enzyme Research Laboratory, IN). After stimulation, cells were
washed, stained and analyzed by flow cytometry.
3
For Amnis (EMD-Millipore, WA) analysis, 10x106 PBMCs were cultured in media or 50
U/ml of thrombin for 15 minutes at 370C. Cells were washed and incubated for 10
minutes with 10 µg/ml human IgG (Sigma-Aldrich, MO) to prevent potential Fc receptor
binding. The cells were then stained with anti-CD3 PE-Cy7 (BD, clone SP34.2); anti-
CD4 PE (BD Pharmingen, clone SK3); anti-CD42b FITC (BD Pharmingen, clone HIP1);
and anti-CD62P P-Selectin APC (BD Pharmingen, clone AK-4). After staining, the cells
were fixed in paraformaldehyde 4% for 10 minutes. Samples were collected on the
Amnis (EMD-Millipore, WA). IDEAS software (EMD-Millipore, WA) was used to
generate the ImageStream gallery display. Image display was generated by setting the
pixel range above threshold background noise. The display pixel upper range was set to
255 for all intensities above this background. The composite views facilitate image
analysis between different channels for the same cell, i.e. platelet-T cell conjugates. For
the overlay images, the contributing contribution between any 2 or more overlaid images
can be varied, in particular to highlight fluorescence in any overlaid channels. The
percent image contributions for each channel in these composite views (Figure 5A and
Supplemental Figure S3C) were set to 100% for brightfield, 30% for CD42b and 35% for
CD62P.
Scanning electron microscopy
Platelet isolation: Platelet rich plasma was obtained by centrifugation at 200g for 20
minutes and diluted in a 1:1 volume with HEP buffer (140 mM NaCl, 2.7 mM KCl, 3.8
mM Hepes, 5 mM EGTA, pH 7.4) followed by centrifugation at 100g for 20 minutes at
room temperature. The supernatant was discarded and the platelet rich pellet was washed
in buffer (10mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% (w/v) dextrose, pH
7.35). The pellet was then resuspended in Tyrode’s buffer (134 mM NaCl, 12 mM
4
NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM Hepes, 3 mg/ml
BSA, 5 mM glucose, pH 7.4). Platelets were counted using a hemacytometer and
adjusted to the desired concentration (20x106 cells/ml) before being added to T cells
suspension as described below.
CD8 T cells and platelets stimulation: CD8 T cells were isolated by negative selection
(Miltenyi Biotec, CA) and stained with anti-CD3 FITC (BD Pharmingen, clone SK7);
anti-CD8 Qdot605 (Invitrogen, clone 3B5); anti-CD45RA Pacific Blue (Invitrogen, clone
MEM-56); anti-CD27 APC-H7 (BD Pharmingen, clone M-T271); and the CD8 T cell
memory subset (CD3+CD8+CD45RA-CD27+) was obtained by FACS sorting. The sorted
CD8 memory T cells were resuspended (at 2x106 cells/ml) in HBSS + Ca++ Mg++ + 1
mg/ml BSA and platelets were added at a 1:1 ratio. The cells were stimulated for 15
minutes at 370C with 50 U/ml of thrombin (Enzyme Research Laboratories, IN), spun
down and washed with PBS. The samples were fixed in a cocktail of 4% formaldehyde
and 2% glutaraldehyde in 0.1M cacodylate buffer and post fixed using a 1% osmium
tetroxide solution. They were then dehydrated in a series of graded alcohols and air dried
after a final dehydration course of tetramethylsilane. Subsequently, the samples were
sputter coated with a thin layer of iridium and imaged utilizing a Hitachi S-4500 field
emission scanning electron microscope (Tokyo, Japan).
Statistical analysis method
A nonparametric unpaired Mann-Whitney test was used to compare the data between the
HIV infected patients and healthy controls. A nonparametric, paired Wilcoxon test was
used to compare unstimulated and thrombin stimulated PBMCs. Statistically significant
5
differences were considered to be those whose p value was <0.05. Spearman's
correlation coefficients were use to evaluate correlations.
6
Supplementary Figures
Figure S1. PSGL1 expression on CD4 and CD8 T cell subsets and recombinant
CD62P binding affinity on activated HLADR+CD38+ T cells
(A) PSGL1 expression on CD4 and CD8 T cells. Flow cytometry gating strategy used to
analyze PSGL1 expression on CD4 and CD8 T cell subsets: naïve (CD45RA+CD27+),
memory (CD45RA-CD27+), memory/effector (CD45RA-CD27-), TEM
(CD45RA+CD27-). (B) Overlay histogram of PSGL1 on the CD4 and CD8 T cells
subsets: naïve (gray) memory (black), memory/effector (red), TEM (blue) and istotype
control (dotted gray line). (C) PSGL1 MFI expression in the subsets in CD4 and CD8 T
cells from healthy controls (black symbols, n=13) and HIV infected patients (red
symbols, n=18). (D) Flow cytometry gating strategy used to analyze HLADR and CD38
expression on total CD4 and CD8 T cells. (E) Representative CD62P-Fc binding plots
(gray) overlaid with the IgG control (blue) of a healthy control and HIV infected patient
on HLADR+CD38+ and HLADR-CD38- CD4 and CD8 T cell populations. (F) Percentage
of rCD62P-Fc binding in CD8 and CD4 T cells in the HLADR+CD38+ and HLADR-
CD38- populations from healthy controls (black symbols, n=13) and HIV infected
patients (red symbols, n=18). Paired Wilcoxon test was used for comparisons between
binding of rCD62P-Fc in the HLADR+CD38+ and HLADR-CD38- populations.
Figure S3. Increased activation of platelet and platelet-leukocyte conjugates HIV
infected patients.
(A) Gating strategy of whole blood from HIV infected patients (n= 20) and healthy
controls HC (n=23) from Figure 2 were analyzed for platelet bound to other leukocytes
types such as CD3 negative lymphocytes and high FCS-SSC (monocytes) and
7
free/unbound platelets. (B) Represents the proportion of platelets bound to monocytes
(left panel) and CD3 negative lymphocytes (middle panel). Right panel shows CD62P+
expression on free/unbound platelets. Mann-Whitney test was used for comparisons
between HIV infected patients and healthy controls. p value <0.05 was considered
significant.
Figure S3. Association between circulating activated platelet and activated platelet-
T cell conjugates in HIV infected patients.
(A) Association between the percentage of circulating (free/unbound) activated platelets
(CD42b+CD62P+) and the proportion of activated platelets bound to CD4 and CD8 T
cells in patients with HIV infection (n= 20). (B) Relationship between serum levels of D-
dimer and circulating activated platelet-CD4 and –CD8 T cell conjugates in patients with
HIV infection (n=20).
Figure S4. Increased activation of platelet and platelet-leukocyte conjugates HIV
infected patients.
HIV infected patients (n= 20) and healthy controls HC1 (n=23) from Figure 2 and
healthy controls (HC2, Supplementary Table S1 and S2) were analyzed for platelet T cell
conjugates. Mann-Whitney test was used for comparisons between HIV infected patients
and healthy controls. p value <0.05 was considered significant.
Figure S5. ImageStream gating Strategy.
Gating strategy of samples shown in Figure 5A. PBMCs from healthy controls (n= 10)
and HIV infected patients (n= 5) were cultured in the presence or absence of thrombin
8
(50 U/ml) for 15 minutes. Cells were stained with CD3, CD4, CD42b and CD62P mAbs
and fixed prior to acquisition in ImageStream (Amnis). (A) For analysis, acquired
samples were pre-gated for those cells in focus followed by a singlets side-scatter gate
and a CD42b+ vs SSC gate was performed to eliminate free/unbound platelets (data not
shown). The lymphocytes were gated using the equivalent to FSC vs SSC followed by
CD3+CD4+ for (CD4 T cells, green) and CD3+CD4- for (CD8 T cells, yellow). In
addition, a gate based on CD62P+CD42b+ (pink) was performed to analyze the platelet-T
cells conjugates formation in unstimulated and after thrombin stimulation. Example of
analysis of 10 platelet-CD4 and –CD8 T cell conjugates cultured in media or after
thrombin stimulation. (B) Overlay of the brightfield morphology, CD62P, and CD42b
channels in10 representative free/unbound platelets in unstimulated and stimulated
conditions (samples showed in Figure 5A).
9
Table S1. HIV infected Patients and Healthy controls
HIV+ Patients (n= 18)
Fig 1
HIV+ Patients (n= 20)
Fig 2, 3, 4
HC1 (n=23)
Fig 2,3,4
HC2 (n=24) Fig S2
HIV RNA (copies/ml)
40 40 N/A N/A
CD4 counts (cells/µ l)
691.5 (605-1068)
454 (309-582)
n/a 585.5 (410-812)
CD8 counts (cells/µ l)
757.5 (534-920)
808 (504-1291)
n/a 424 (288-517)
% CD4 HLA-DR+ 38+
5.0 (3.8-7.0)
5.5 (4.3-10.5)
n/a 4.0 (2.3-4.8)
% CD8 HLA-DR+ 38+
15.5 (12.0-22.3)
17 (10.0-22.8)
n/a 8.5 (6.0-11.0)
cART (months)
65.0 (44.8-135.3)
85.5 (15.5-135)
N/A N/A
Aspirin Warfarin (months)
27* (25-92)
34* (7-77.5) 115**
n/a n/a
D-Dimer (ng/ml)
n/d 451 (259-884)
351 (262.3-665.5)
718 (355.3-947.7)
sCD62P (ng/ml)
n/d 291.3 (218-359)
280 (198-315)
160 (114.3-240)
Values are presented as medians (IQR: 25% and 75%) N/A: not applicable, n/a: not available, n/d: not determined Patients on *Aspirin (n=3), **warfarin (n=1)
10
Table S2. Comparisons of marker of activation and activated platelets %Free/unbound
Platelets CD42b+CD62P+
R, p value
%(CD42b+CD62P+) Platelet-
CD4 T cell conjugate R, p value
%(CD42b+CD62P+) Platelet-
CD8 T cell conjugate R, p value
HIV (n=20) %HLADR+CD38+
CD4 T cells
-0.01, 0.990
(0.01, 0.986)*
0.05, 0.820
(0.29, 0.251)*
0.08, 0.720
(0.33, 0.187)* HIV (n=20)
%HLADR+CD38+ CD8 T cells
-0.08, 0.710
(0.02, 0.942)*
0.18, 0.440
(0.39, 0.110)*
0.21, 0.540
(0.40, 0.107)* HIV (n=20) CD4 count
cell/µ l
-0.10, 0.656
(-0.12, 0.622)*
0.04, 0.855
(-0.16, 0.528)*
-0.10, 0.654
(-0.21, 0.416)* HIV (n=20) CD8 count
cell/µ l
0.13, 0.571
(0.06, 0.807)*
-0.08, 0.709
(-0.20, 0.422)
-0.16, 0.485
(-0.24, 0.351) HIV (n=20)
D-dimer ng/ml
0.51, 0.026
(0.68, 0.007)*
0.56, 0.015
(0.68, 0.007)*
0.52, 0.023
(0.62, 0.013)* HIV (n=20)
sCD62P ng/ml
0.30, 0.229
(0.37, 0.157)*
0.39, 0.117
(0.40, 0.121)*
0.22, 0.389
(0.23, 0.386)*
HC1 (n=23) D-dimer
ng/ml
-0.20, 0.473
-0.30, 0.280
-0.30, 0.287
HC1 (n=23)
sCD62P ng/ml
0.01, 0.973
0.29, 0.354
0.26, 0.404
HC2 (n=24) %HLADR+CD38+
CD4 T cells
-0.01, 0.958
-0.07, 0.743
0.01, 0.965
HC2 (n=24)
%HLADR+CD38+ CD8 T cells
0.31, 0.128
-0.08, 0.701
-0.01, 0.928
HC2 (n=24) CD4 count
cell/µ l
0.14, 0.512
0.07, 0.742
-0.03, 0.875
HC2 (n=24) CD8 count
cell/µ l
-0.01, 0.938
0.37, 0.069
0.37, 0.069
HC2 (n=24)
D-dimer ng/ml
0.16, 0.430
-0.04, 0.837
-0.02, 0.900
HC2 (n=24)
sCD62P ng/ml
-0.11, 0.582
0.36, 0.070
0.42, 0.070
*Correlation after removing 3 patients on Aspirin
11
Table S3. cART regime (months)
Patient Number
Months VL <40
ABC/3TC ATV DRV
EFV/ FTC/ TDF
FTC/ TDF
EVG/ COBI TDF/ FTC
ETR 3TC MVC NVP RAL RTV TMP-SMX TER
Figure 2 - Pt 1 282 42
212 54 171 130 212
Figure 2 - Pt 2 140 45
140
Figure 2 - Pt 3 84 84
Figure 2 - Pt 4 102 94 102
94
Figure 2 - Pt 5 72 72 72
72
Figure 2 - Pt 6 145 22 22
145
Figure 2 - Pt 7 117
101
117
Figure 2 - Pt 8 29 29
Figure 2,3,4 - Pt 9 9 9
Figure 2,3,4 - Pt 10 6 5
Figure 2,3,4 - Pt 11 47 47
47
Figure 2,3,4 - Pt 12 120 120 103
120
Figure 2,3,4 - Pt 13 87 4
4
4
Figure 2,3,4 - Pt 14 4
Figure 2,3,4 - Pt 15 8 8
Figure 2,3,4 - Pt 16 11 11 11
11
Figure 2 - Pt 17 293 106 51
56 293
Figure 2 - Pt 18 100 4 100
Figure 2 - Pt 19 42 42
42
Figure 2 - Pt 20 174 80 80
43 80 174
ABC: abacavir; 3TC: lamivudine; ATV: atazanavir; COBI: Cobicistat; EFV: efavirenz; EVG: Elvitegravir; FTC: emtricitabine; ETR: etravirine; DRV: darunavir; MVC: maraviroc; NVP: nevirapine; RAL: raltegravir; RTV: ritonavir; TDF: tenofovir; TER: Terbinafine; TMP-SMX: Trimethoprim-sulfamethoxaz ABC: abacavir; 3TC: lamivudine; ATV: atazanavir; COBI: Cobicistat; EFV: efavirenz; EVG: Elvitegravir; FTC: emtricitabine; ETR: etravirine; DRV: darunavir; MVC: maraviroc; NVP: nevirapine; RAL: raltegravir; RTV: ritonavir; TDF: tenofovir;; TER: Terbinafine; TMP-SMX: Trimethoprim-sulfamethoxaz
12
Table S4. Associated complication Patient Number AD/CAD/PPx DEPR Dia HSV HLD/HTN/HYTG HYT Other
Figure 2 - Pt 1 AD: Warfarin (115)
Acyclovir (282)
HLD: Atorvastatin (172) HTN: Lisinopril (147)
Figure 2 - Pt 2 PPx: Asprin (57) Melatonin (92)
Figure 2 - Pt 3 Metformin (128)
HTN: Lisinopril (90)
Figure 2 - Pt 4 Clonazapam (150)
Figure 2 - Pt 5
Figure 2 - Pt 6
Figure 2 - Pt 7 Citalopram (158);
HLD: Atorvastatin (140)
Levothyroxine (56)
PD: Carbidopa-levodopa-entacapone (46); Lidocaine transdermal patch (64); Ropinirole (67)
Figure 2 - Pt 8
Figure 2,3,4 - Pt 9
Valacyclovir (26)
Figure 2,3,4 - Pt 10
Figure 2,3,4 - Pt 11
PPx: Asprin (84)
Valacyclovir (142) HLD: Atorvastatin
(132)
Figure 2,3,4 - Pt 12 Bupropion (100)
HLD: Atorvastatin (39)
Levothyroxine (187)
CS: Diphenhydramine (44); Fexofenadine (55)
Figure 2,3,4 - Pt 13
Figure 2,3,4 - Pt 14 Citalopram (16);
Zolpidem (16)
GERD: Omeprazole (8)
Figure 2,3,4 - Pt 15
PSO: Hydrocortisone (12)
Figure 2,3,4 - Pt 16
Valacyclovir (89)
HYP: Potassium phosphate (47)
Figure 2 - Pt 17
Figure 2 - Pt 18
Metformin (12)
HYTG: Atorvastatin (17)
OP: Calcium carbonate (125); Cholecalciferol (71)
Figure 2 - Pt 19
HTN: Lisinopril (264)
C.Tox: Clinadmycin (376); Dapsone (748); leucovorin (44); Pyrimethamine (34) SzD: Levetiracetam (44); Phenytoin (44)
Figure 2 - Pt 20
CAD: Brilinta (2) PPx: Asprin (66)
Metformin (153)
HYTG: Pravastatin (101)
Months of treatment is indicated in parenthesis. Abreviations: AD: Atrial Dysrythmia; CAD: Coronary Artery Disease; PPx: Prophylaxis; C.Tox.: Cerebral Toxoplasmosis; CS: Chronic Sinusitis; DEPR: Depression; Dia: Diabetes; GERD: Gastroesophageal reflux disease; HSV: Herpes Simplex Virus; HYP: Hypophosphatemia; HLD: hyperlipidemia; HTN: Hypertension; HYT: Hypothyroidism; HYTG: Hypertriglyceridemia; PD: Parkinson’s Disease; PSO: Psoriasis; OP: Osteoporosis; SzD: Seizure Disorder.
SSC
SSC
CD
8
CD
27
FSC FSC-W CD3
CD8 T Cell
CD45RA
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC-A
0 50K 100K 150K 200K 250KFSC-W
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105
<FITC-A>: CD3
0
102
103
104
105
<Qdo
t 605
-A>:
Cd8
0 102 103 104 105
<Pacific Blue-A>: CD45RA
0
102
103
104
105
<AP
C-C
y7-A
>: C
D27
0 102 103 104 105
<Pacific Blue-A>: CD45RA
0
102
103
104
105
<AP
C-C
y7-A
>: C
D27
TEM Memory/ Effector
Memory/ Effector
Memory Naive Memory Naive
CD4 T Cell
CD4
CD8
Healthy Control HIV+
CD4 T Cells
Naive C
D4
MFI PSGL1
HC Naive C
D4
MFI PSGL1
HIVMem
ory CD4
MFI PSGL1
HC Memory
CD4
MFI PSGL1
HIV
Effecto
r/Mem
ory CD4
MFI PSGL1
HC
Effecto
r/Mem
ory CD4
MFI PSGL1
HIV
0
10000
20000
30000
40000
50000
PSGL1 MFI CD4
5
4
3
2
1
0
Naive Memory Memory/ Effector
HIV+ (n=18) HC (n=13)
0 102 103 104 105
<APC-A>
0
20
40
60
80
100
% of M
ax
0 102 103 104 105
<APC-A>
0
20
40
60
80
100% o
f Max
0 102 103 104 105
<APC-A>
0
20
40
60
80
100
% of M
ax
0 102 103 104 105
<APC-A>
0
20
40
60
80
100
% of M
ax
PSG
L1 M
FI (x
104 )
Naive C
D8
MFI PSGL1
HCNaive C
D8
MFI PSGL1
HIVMem
ory CD8
MFI PSGL1
HCMemory
CD8
MFI PSGL1
HIV
Effecto
r/Mem
ory CD8
MFI PSGL1
HC
Effecto
r/Mem
ory CD8
MFI PSGL1
HIV
TEM CD8
MFI PSGL1
HC TEM CD8
MFI PSGL1
HIV
0
10000
20000
30000
40000
50000
PSGL1 MFI CD8
Naive Memory Memory/ Effector
TEM
5
4
3
2
1
0
CD8 T Cells
CD
4 T
Cel
ls
CD
8 T
Cel
ls
Naive
Memory Memory/Effector TEM
mIgG control
(A)
(B) (C)
PSGL1
Supplementary Figure S1
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
Healthy Control
CD
4 T
Cel
ls
CD
8 T
Cel
ls
HIV+
SSC
CD62P
(D)
HIV+ (n=18) HC (n=13)
% T
cel
l CD
62P-
Fc+
Total CD4 H
LA-DR+/CD38
+ %rC
D62P+
HC
Total CD4 H
LA-DR+/CD38
+ %rC
D62P+
HIV
Total C
D4 HLA-D
R-/CD38
- %rC
D62P+
HC
Total C
D4 HLA-D
R-/CD38
- %rC
D62P+
HIV
0
20
40
60
80
100
CD4 Total CD38/HLA-DR then % CD62P+
100
80
60
40
20
0
CD4 T Cells
CD8 T Cells
Total CD8 H
LA-DR+/CD38
+ %rC
D62P+
HC
Total CD8 H
LA-DR+/CD38
+ %rC
D62P+
HIV
Total C
D8 HLA-D
R-/CD38
- %rC
D62P+
HC
Total C
D8 HLA-D
R-/CD38
- %rC
D62P+
HIV
0
20
40
60
80
100
CD8 Total CD38/HLA-DR then % CD62P+
100
80
60
40
20
0 HLADR+
CD38+ HLADR-
CD38-
p<0.001
p<0.001
p=0.001
p<0.001
22.3 77.6 44.4 52.5
39.3 60 12 87.9 33.8 65.7
44.2 54.9
19.7 80.1
25.6 74.3
SSC
SSC
CD
8
FSC FSC-W CD3
CD8 T Cell
HLADR 0 50K 100K 150K 200K 250K
FSC-A
0
50K
100K
150K
200K
250K
SSC-A
0 50K 100K 150K 200K 250KFSC-W
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105
<FITC-A>: CD3
0
102
103
104
105
<Qdo
t 605
-A>:
Cd8
CD4 T Cell
CD4
CD8
CD
38
0 102 103 104 105
<PE-A>: HLA-DR
0
102
103
104
105
<Per
CP
-A>:
CD
38
0 102 103 104 105
<PE-A>: HLA-DR
0
102
103
104
105
<Per
CP
-A>:
CD
38
0.46 57.6
0.52 41.4
0.77 74
1.24 24
HLADR-
CD38- HLADR+
CD38+
(E) (F)
HLADR-
CD38- HLADR+
CD38+
0 102 103 104 105<PerCP-Cy5-5-A>
0
102
103
104
105
<APC-A>
0 102 103 104 105<PerCP-Cy5-5-A>
0
102
103
104
105
<APC-A>
0 102 103 104 105<PerCP-Cy5-5-A>
0
102
103
104
105
<APC-A>
0 102 103 104 105<PerCP-Cy5-5-A>
0
102
103
104
105
<APC-A>
0 102 103 104 105<PerCP-Cy5-5-A>
0
102
103
104
105
<APC-A>
0 102 103 104 105<PerCP-Cy5-5-A>
0
102
103
104
105
<APC-A>
0 102 103 104 105
<FITC-A>
0
102
103
104
105
<Qdo
t 605
-A>
0 50K 100K 150K 200K 250KFSC-W
0
50K
100K
150K
200K
250K
SSC-A
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC-A
0 102 103 104 105<PerCP-Cy5-5-A>
0
50K
100K
150K
200K
250K
SSC-A
7.23 11.8
70.7 10.3
Healthy Controls
HIV+ (n=20) HC (n=23)
1.12 6.58
3.26 89.1
R2 (free/unbound Platelets)
L (CD3 nega6ve lymphocytes) 2.13 2.07
6.59 89.2
CD42b FSC
SSC
SSC
CD
8
R1 R2
R1
M (Monocytes)
HIV+ patients
M L
Supplementary Figure S2
L (CD3 nega6ve lymphocytes)
SSC
FSC-W CD3
6.27 19.1
11 63.7
4.59 6.19
80.3 8.89
0.068 20.8
78.9 0.263
(A)
(B)
CD3-
CD62P+/CD42
b- ND
CD3-
CD62P+/CD42
b- HIV+
CD3-
CD62P+/CD42
b+ ND
CD3-
CD62P+/CD42
b+ HIV+
CD3-
CD62P-/C
D42b+ ND
CD3-
CD62P-/C
D42b+ HIV+
0
5
10
15
20
25
Quadrants CD3-
% P
ositi
ve
CD
62P+
/CD
42b+
ND
CD
62P+
/CD
42b+
HIV CD
62P-
/CD
42b+
ND
CD
62P-
/CD
42b+
HIV
0
20
40
60
80
100
Perc
ent (
%)
Free Platelets
CD
62P
+/C
D42
b-N
D
CD
62P
+/C
D42
b-H
IV+
CD
62P
+/C
D42
b+N
D
CD
62P
+/C
D42
b+H
IV CD
62P
-/CD
42b+
ND
CD
62P
-/CD
42b+
HIV
0
10
20
30
40
Per
cent
(%)
Monos
0 20
40
60
80
100
p=0.017
p=0.019
0
20
40
30
10
% p
late
let-l
euko
cyte
co
njug
ates
p=0.016
0
10
20
15
5
25
CD42b-/ CD62P+
CD42b+/ CD62P+
CD42b+/ CD62P-
CD42b+/ CD62P+
CD42b+/ CD62P-
p=0.032
ns
p=0.041 ns
ns
R2 (free/unbound Platelets) L (CD3 nega6ve lymphocytes) M (Monocytes)
CD42b-/ CD62P+
CD42b+/ CD62P+
CD42b+/ CD62P-
CD
62P
CD42b
Supplementary Figure S3
(A)
% CD42b+CD62P+
(Free/unbound)
% P
late
let (
CD
42b+
CD
62P+
) -C
D4
T ce
ll co
njug
ate
0
4
6
2
8
0 10 20 30 40 500
2
4
6
8
HIV CD4 TOTAL CD62P+/CD42b+
0 10 20 30 40 50 0
4
6
2
8
0 10 20 30 40 500
2
4
6
8
HIV CD8 TOTAL CD62P+/CD42b+
0 10 20 30 40 50
R= 0.65 p= 0.005
D-dimer (ng/ml)
(B)
D-dimer (ng/ml)
0 500 1000 1500 2000 25000
2
4
6
8
HIV CD4 TOTAL CD62P+/CD42b+
0 500 1000 1500 2000 2500 0
4
6
2
8
0 500 1000 1500 2000 25000
2
4
6
8
HIV CD8 TOTAL CD62P+/CD42b+
0 500 1000 1500 2000 2500 0
4
6
2
8
% P
late
let (
CD
42b+
CD
62P+
) -C
D8
T ce
ll co
njug
ate
% P
late
let (
CD
42b+
CD
62P+
) -C
D4
T ce
ll co
njug
ate
% P
late
let (
CD
42b+
CD
62P+
) -C
D8
T ce
ll co
njug
ate
R= 0.52 p= 0.023
R= 0.66 p= 0.004
R= 0.56 p= 0.015
% CD42b+CD62P+
(Free/unbound)
Supplementary Figure S4
HC CD4T
OTAL CD62
P+/CD42
b-
HC2 Total
CD4
CD42b-/C
D62P+
HC-2
HIV CD4 T
OTAL CD62
P+/CD42
b-
HC CD4 T
OTAL CD62
P+/CD42
b+
HC2 Total
CD4
CD42b+/C
D62P+
HC-2
HIV CD4 T
OTAL CD62
P+/CD42
b+
HC CD4 T
OTAL CD62
P-/CD42
b+
HC2 Total
CD4
CD42b+/C
D62P-
HC-2
HIV CD4 T
OTAL CD62
P-/CD42
b+0
5
10
15
20
25
% c
onju
gate
s
Total CD4
HC CD8 T
OTAL CD62
P+/CD42
b-
HC2 Total
CD8 C
D42b-/C
D62P+
HC-2
HIV CD8 T
OTAL CD62
P+/CD42
b-
HC CD8 T
OTAL CD62
P+/CD42
b+
HC2 Total
CD8 C
D42b+/C
D62P+
HC-2
HIV CD8 T
OTAL CD62
P+/CD42
b+
HC CD8 T
OTAL CD62
P-/CD42
b+
HC2 Total
CD8 C
D42b+/C
D62P-
HC-2
HIV CD8 T
OTAL CD62
P-/CD42
b+0
5
10
15
20
25
% c
onju
gate
s
Total CD8%
CD
4 T
cell
bi
ndin
g pl
atel
ets
% C
D8
T ce
ll
bind
ing
plat
elet
s
0 5
10
15
20
25
0 5
10
15
20
25
CD42b-/ CD62P+
CD42b+/ CD62P+
CD42b+/ CD62P-
CD42b-/ CD62P+
CD42b+/ CD62P+
CD42b+/ CD62P-
p<0.001
p<0.001 ns
p=0.002
ns p=0.004
ns
ns p=0.016 p<0.001
p=0.019 ns p=0.003
ns p=0.003
ns
p=0.033 p=0.009
Healthy Control (HC1, n=23) Healthy Control (HC2, n=24)
HIV+ (n=20)
Supplementary Figure S5
Free/Unbound Platelets
CD42b CD62P Overlay CD42b CD62P Overlay CD42b CD62P Overlay CD42b CD62P Overlay
Healthy Control HIV+
Media Thrombin Media Thrombin
Healthy Control HIV+ (A)
(B)