PS + MPs PS - MPs 37% 36% 64% 64%
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1 Supplementary Figure 1. Amount and distribution of phosphatidylserine negative (PS - ) and phosphatidylserine positive (PS + ) MPs in 280 SLE patients and 280 controls. Circles are proportional to the total number of MPs among SLE patients and controls. SLE patients had roughly 6 times more total MPs (i.e. PS + + PS - MPs) than controls; 3 times more PS + MPs and 10 times more PS - MPs. SLE Patients Controls PS + MPs PS - MPs 64% 37% 36% 64% Circles are proportional to the total number of MPs among SLE patients and controls. SLE patients had roughly 6 times more total MPs (i.e. PS + + PS - MPs) than controls; 3 times more PS + MPs and 10 times more PS - MPs. 1
2 Supplementary Figure 2. Flow cytometric analysis of microparticles. The microparticle (MP) gate was determined using Megamix beads (0.5, 0.9 and 3.0 µm beads) (A). Conjugate isotype-matched immunoglobulins with no reactivity against human antigens were used as negative controls (B). MPs were defined as particles less than 1.0 µm in size (the MP-gate in panel A) and positive or negative to lactadherin (C), i.e. PS - and PS + MPs. Each subpopulation was further phenotyped according to origin and expression of inflammation and activation markers. 2
3 Supplementary Table 1. Demographics, SLE criteria and their relationship to phosphatidylserine negative MPs in 280 SLE patients. PS - MPs PMPs PMPs CD40L EMPs EMPs VCAM EMPs TF LMPs LMPs TF MMPs HMGB1 PMPs C4d EMPs C4d Age (n=280) (-) <0.05 Gender Female (n=280) (+) < *** (+) <0.001 * Disease duration (n=280) (+) <0.01 (+) <0.05 Smoking Current (n=280) (+) <0.01 (+) <0.01 (+) <0.001* LUPUS MANIFESTATIONS Butterfly (n=280) (+) <0.05 (+) <0.05 Discoid (n=280) Photosensitivity (n=280) Oral ulcers (n=280) Arthritis (n=280) (-) <0.01 Pleuritis (n=280) (+) <0.05 (+) < ** (+) <0.05 Pericarditis (n=280) Serositis (n=280) (+) <0.05 (+) < * (+) <0.05 (+) <0.05 Nephritis (n=280) (-) <0.05 (-) <0.05 Psychosis (n=280) Seizures (n=280) Leucopenia (n=280) Lymphopenia (n=280) (-) <0.05 Thrombocytopenia (n=280) (+) <0.05 Hemolytic anemia (n=280) adna, ever (n=280) (+) <0.05 (+) <0.05 (+) <0.01 asm, ever (n=280) (-) <0.05 Immunological criteria (n=280) (+) <0.05 (+) <0.05 (-) <0.05 (+) <0.05 SLAM >6 (n=277) AUTOANTIBODIES AT INCLUSION dsdna (n=279) (+) <0.01 Nucleosomes (n=280) (+) <0.05 (-) <0.01 Sm (n=280) (-) <0.05 RNP68 (n=280) SSA (n=280) SSB (n=280) (+) <0.01 (+) <0.05 3
4 Lupus anticoagulant (n=280) (-) <0.05 Cardiolipin IgG (n=262) (+) <0.05 (+) <0.05 (+) <0.05 Cardiolipin IgM (n=262) (+) <0.05 (+) <0.05 (+) <0.05 Cardiolipin IgA (n=262) (+) <0.05 (+) <0.05 (+) <0.05 β 2GP-1 IgG (n=262) (+) <0.05 (+) <0.05 (+) <0.05 (+) <0.05 β 2GP-1 IgM (n=262) (+) <0.05 (+) <0.05 (+) <0.05 β 2GP-1 IgA (n=262) (+) <0.05 (+) <0.05 (+) <0.05 (+) <0.05 INFLAMMATION hs CRP (n=274) (-) <0.05 (-) <0.05 C3 (n=264) (-) <0.05 (-) <0.001 (+) <0.05 (-) <0.01 (+) <0.05 C4 (n=264) (-) <0.05 (+) <0.05 (-) <0.001 (+) <0.05 Fibrinogen (n=271) (-) <0.05 (-) <0.05 (+) <0.001 Creatinine (n=277) (-) <0.05 (-) <0.05 (+) <0.05 Cystatin C GFR (n=271) (+) < *** (+) <0.001 * (-) <0.01 IL-6 (n=207) TNF-α (n=207) (-) <0.001 * (-) <0.05 TNFR1 (n=275) (-) <0.001 ** (-) <0.001 * (+) <0.05 TNFR2 (n=276) (-) <0.001 * (-) <0.05 (+) <0.01 (-) <0.05 IP-10 (n=274) (+) <0.01 (-) <0.01 (-) <0.05 MCP-1 (n=275) (+) < *** (+) <0.05 OTHER PARAMETERS APS (n=280) Any arterial event (n=280) (+) <0.05 Any vascular event (n=280) (-) <0.05 VTE (n=280) Values are given as direction (+/-) and p-value of association (t-test) for nominal/ordinal data. Blank = non significant (p-value>0.05). The p- values that were still significant after adjustment according to Bonferroni are labeled with asterisk: *** p<0.001, ** p<0.01, * p<0.05. adna = anti-dna antibody; asm = Anti-Smith antibodies; SLAM = Systemic Lupus Activity Measure 16 ; dsdna = doublestranded DNA; ; Sm = Smith; RNP = ribonucleoprotein; SSA = Sjogrens syndrome antigen A; SSB = Sjogrens syndrome antigen B; β 2 GP-1 = beta 2 glykoprotein-1; hscrp = high sensitivity C reactive protein; C3/C4 = Complement factor 3/4; GFR = GFR - glomerular filtration rate; IL-6 = interleukin 6; TNF = Tumor necrosis factor; TNFR = Tumor necrosis factor receptor; IP10 = Interferon gamma-induced protein 10; MCP-1 = Monocyte Chemoattractant Protein-1; APS = Antiphospholipid syndrome, according to Miyakis et al 53 ; VTE = Venous thromboembolism. 4
5 Supplementary Table 2. Demographics, SLE criteria and their relationship to phosphatidylserine positive MPs in 280 SLE patients. PS + MPs PMPs PMPs CD40L EMPs EMPs VCAM EMPs TF LMPs LMPs TF MMPs HMGB1 PMPs C4d EMPs C4d Age (n=280) (-) <0.01 (+) <0.05 Gender Female (n=280) Disease duration (n=280) (-) <0.05 (+) <0.05 (+) <0.001 * (+) <0.05 Smoking Current (n=280) (-) <0.05 (-) <0.05 (+) <0.001* LUPUS MANIFESTATIONS Butterfly (n=280) (+) <0.05 Discoid (n=280) Photosensitivity (n=280) (-) <0.05 Oral ulcers (n=280) Arthritis (n=280) (+) <0.05 Pleuritis (n=280) (-) <0.05 Pericarditis (n=280) Serositis (n=280) (-) <0.05 (+) <0.05 Nephritis (n=280) (-) <0.05 Psychosis (n=280) (+) <0.05 Seizures (n=280) (-) <0.05 Leucopenia (n=280) Lymphopenia (n=280) (+) <0.05 (+) <0.05 Thrombocytopenia (n=280) Hemolytic anemia (n=280) adna, ever (n=280) asm, ever (n=280) (+) <0.05 Immunological criteria (n=280) SLAM >6 (n=277) AUTOANTIBODIES AT INCLUSION dsdna (n=279) (-) <0.05 (+) <0.05 Nucleosomes (n=280) Sm (n=280) RNP68 (n=280) SSA (n=280) SSB (n=280) Lupus anticoagulant (n=280) Cardiolipin IgG (n=262) (-) <0.05 (-) <0.05 Cardiolipin IgM (n=262) (-) <0.01 (+) <0.05 5
6 Cardiolipin IgA (n=262) β 2GP-1 IgG (n=262) β 2GP-1 IgM (n=262) β 2GP-1 IgA (n=262) (+) <0.05 (-) <0.05 (-) <0.01 INFLAMMATION MARKERS (-) <0.05 hs CRP (n=274) C3 (n=264) (+) <0.05 (+) <0.05 C4 (n=264) (+) <0.05 Fibrinogen (n=271) Creatinine (n=277) Cystatin C GFR (n=271) (+) <0.05 IL-6 (n=207) TNF-α (n=207) TNFR1 (n=275) TNFR2 (n=276) IP-10 (n=274) (+) <0.05 MCP-1 (n=275) (-) <0.05 OTHER PARAMETERS (-) <0.05 APS (n=280) (+) <0.05 (-) <0.05 Any arterial event (n=280) (-) <0.05 Any vascular event (n=280) (+) <0.01 VTE (n=280) Values are given as direction (+/-) and p-value of association (t-test) for nominal/ordinal data. Blank = non significant (p-value>0.05). The p-values that were still significant after adjustment according to Bonferroni are labeled with asterisk: *** p<0.001, ** p<0.01, * p<0.05. adna = anti-dna antibody; asm = Anti-Smith antibodies; SLAM = Systemic Lupus Activity Measure 16 ; dsdna = doublestranded DNA; ; Sm = Smith; RNP = ribonucleoprotein; SSA = Sjogrens syndrome antigen A; SSB = Sjogrens syndrome antigen B; β 2 GP-1 = beta 2 glykoprotein-1; hscrp = high sensitivity C reactive protein; C3/C4 = Complement factor 3/4; GFR = GFR - glomerular filtration rate; IL-6 = interleukin 6; TNF = Tumor necrosis factor; TNFR = Tumor necrosis factor receptor; IP10 = Interferon gamma-induced protein 10; MCP-1 = Monocyte Chemoattractant Protein-1; APS = Antiphospholipid syndrome, according to Miyakis et al 53 ; VTE = Venous thromboembolis 6
7 Supplementary Table 3. Demographics and their relationship to phosphatidylserine negative microparticles in 280 controls PS - MPs PMP PMP CD40 L EMP EMP VCAM EMP TF LMP LMP TF MMP HMGB1 PMP C4d EMP C4d Age (n=280) (-) 0.05 Gender Female (n=280) (+) 0.05 Smoking Current (n=280) (+) 0.05 (+) 0.05 (+) 0.05 INFLAMMATION MARKERS hs CRP (n=279) Fibrinogen (n=266) (+) 0.05 (+) 0.05 Creatinine (n=275) (+) 0.05 (+) 0.05 Cystatin C GFR (n=264) (-) 0.05 (-) 0.05 TNFR1 (n=274) (+) 0.05 TNFR2 (n=274) IP-10 (n=271) (+) 0.05 (+) 0.05 (+) 0.05 (+) 0.05 MCP-1 (n=271) Values are given as direction (+/-) and p-value of association (t-test) for nominal/ordinal data. Blank = non significant (p-value>0.05). The p- values that were still significant after adjustment according to Bonferroni are labeled with asterisk: *** p<0.001, ** p<0.01, * p<0.05. hscrp = high sensitivity C reactive protein; GFR = GFR - glomerular filtration rate; IL-6 = interleukin 6; TNF = Tumor necrosis factor; TNFR = Tumor necrosis factor receptor; IP10 = Interferon gamma-induced protein 10; MCP-1 = Monocyte Chemoattractant Protein-1. 7
8 Supplementary Table 4. Demographics and their relationship to phosphatidylserine positive microparticles in 280 controls Age (n=280) PS+ MPs PMPs PMPs CD40 L EMPs EMPs VCAM EMPs TF LMPs LMP TF MMPs HMGB1 PMPs C4d EMPs C4d Gender Female (n=280) (+) 0.05 (+) 0.05 Smoking Current (n=280) (+) 0.05 (+) 0.05 (+) 0.05 INFLAMMATION MARKERS hs CRP (n=279) Fibrinogen (n=266) (+) 0.05 (+) 0.05 * (+) 0.05 * (+) 0.05 (+) 0.05 Creatinine (n=275) (+) 0.05 (+) 0.05 Cystatin C GFR (n=264) (-) 0.05 (-) 0.05 TNFR1 (n=274) (+) 0.05 * (+) 0.05 TNFR2 (n=274) (+) 0.05 IP-10 (n=271) (+) 0.05 (+) 0.05 MCP-1 (n=271) (+) 0.05 (+) 0.05 (+) ** (+)0.001 ** (+) ** Values are given as direction (+/-) and p-value of association (t-test) for nominal/ordinal data. Blank = non significant (p-value>0.05). The p- values that were still significant after adjustment according to Bonferroni are labeled with asterisk: *** p<0.001, ** p<0.01, * p<0.05. hscrp = high sensitivity C reactive protein; GFR = GFR - glomerular filtration rate; IL-6 = interleukin 6; TNF = Tumor necrosis factor; TNFR = Tumor necrosis factor receptor; IP10 = Interferon gamma-induced protein 10; MCP-1 = Monocyte Chemoattractant Protein-1. 8
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