Supplementary Material and Methods Patients and healthy

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


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 14 Citalopram (16);

Zolpidem (16)

GERD: Omeprazole (8)


PSO: Hydrocortisone (12)


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


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


(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


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


0 500 1000 1500 2000 2500 0

4

6

2

8

0 500 1000 1500 2000 25000

2

4

6

8


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)


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)


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)

Documents

Supplementary Material and Methods Patients and healthy