PMC

Platelet count in anti-P-selectin autoantibodies-positive/negative patients with ITP and pSS. (A) Platelet count in P-selectin autoantibodies-positive/negative patients with ITP and pSS. There was a lower platelet count in P-selectin autoantibodies-positive patients than in P-selectin autoantibodies-negative patients (P=0.002). (B) Platelet count in P-selectin autoantibodies-positive/negative ITP patients and pSS patients with thrombocytopenia. There was a lower platelet count in P-selectin autoantibodies-positive patients than in P-selectin autoantibodies-negative patients (P=0.000) among ITP patients and pSS patients with thrombocytopenia. (C) Platelet count in P-selectin autoantibodies-positive/negative pSS patients. There was a lower platelet count in P-selectin autoantibodies-positive pSS patients than in P-selectin autoantibodies-negative pSS patients (P=0.002). (D) Platelet count in P-selectin autoantibodies-positive/negative pSS patients with thrombocytopenia. There was a lower platelet count in P-selectin autoantibodies-positive patients than in anti-P-selectin autoantibodies-negative patients (P=0.012).

Analysis of the extent of cell rolling (A,C,G,H) and firm (B,D–H) adhesion on substrates uniformly functionalized with P-selectin and/or ICAM-1 in parallel plate flow chamber-based adhesion assays. The extent of rolling adhesion on P-selectin (A) and P-selectin + ICAM-1 (C) is inversely proportional to wall shear stress (T_wall) and P-selectin concentration while the extent of firm adhesion on both P-selectin (B) and P-selectin+ICAM-1 (D) decreases with increasing wall shear stress but increases with increasing P-selectin concentration. ICAM-1 alone does not support substantial levels of rolling (A) or firm (B) adhesion, and ICAM-1 concentration has little to no effect on the extent of firm adhesion when co-presented with P-selectin (E). (F) Differences in levels of firm adhesion on P-selectin + ICAM-1 versus P-selectin alone were significantly non-zero only when the P-selectin concentration was 2.5 μg/mL or greater. (G) The proportion of firmly adhered of total interacting cells is higher when P-selectin was co-presented with ICAM-1 than when P-selectin was singly presented at low wall shear stresses and intermediate and high P-selectin concentrations (2.5, 10 μg/mL). (H) The ratio of firm to rolling adhesion is higher when P-selectin and ICAM-1 are co-presented compared to P-selectin alone and greatest at low wall shear stresses and intermediate to higher P-selectin concentrations. Dashed line represents an equal extent of firm to rolling adhesion. (A–H) Data represent 6 sampled imaging fields of view from 3 independently run experiments, with mean ± SEM indicated for each condition. (A–D) Two-way ANOVA with Bonferroni correction for multiple comparisons, † and § indicate significance of comparisons against lowest wall shear stress and lowest P-selectin or ICAM-1 concentration, respectively. One-sample t-tests were conducted for all data, where Ho=0 (A–F) or Ho=1 (H), and ^* indicates significantly non-zero or non-unity means, respectively. (G) One-way ANOVA with Bonferroni correction for multiple comparisons, (H) t-tests between P and P+I for each condition, (G–H) $ indicates significance of comparisons between P and P+I. (A–H) 0.5, 1.0, 1.5, 2.5, and 10 in legends and axis titles indicate concentrations of either P-selectin (P) or ICAM-1 (I) in μg/mL.

Analysis of circulating soluble P-selectin. Soluble P-selectin levels in plasma generated from untreated (black bars) and 21-day nephritic (white bars) Pt⁺/EC⁺, Pt⁺/EC^–, Pt^–/EC⁺, and Pt^–/EC^– mice were assessed (a), and soluble P-selectin from plasma of mice was characterized by Western blot (b). (a) Levels of soluble P-selectin in plasma were evaluated by an ELISA assay. OD readings from samples obtained from P-selectin–deficient mice (Pt^–/EC^–) served as the negative control. All four sets of untreated mice had minimal levels of soluble P-selectin. Nephritic wild-type animals (Pt⁺/EC⁺) had increased levels compared with untreated mice, as did mice with P-selectin expression on endothelial cells but not on platelets (Pt^–/EC⁺). Mice expressing P-selectin only on platelets (Pt⁺/EC^–) had levels of soluble P-selectin that were not significantly higher than those in the negative control (Pt^–/EC^–). *P < 0.005 compared with Pt⁺/EC⁺ and Pt^–/EC⁺. n = 4 per each group. (b) Western blot analysis of P-selectin in platelets and plasma. Platelet lysates and P-selectin immunoprecipitates from plasma samples of a wild-type and P-selectin–null mouse were reduced and electrophoresed on a 6% polyacrylamide gel, followed by Western blotting using a polyclonal anti–P-selectin antibody. P-selectin in plasma of a normal and a 21-day nephritic wild-type mouse (lanes 1 and 4, respectively) was present as a doublet and had an increased electrophoretic mobility (85–87 kDa) compared with platelet P-selectin (132 kDa) (lane 2). This suggests that plasma P-selectin is a shed form of the transmembrane-bound receptor. No P-selectin was detected in platelets of P-selectin–null mice or in plasma of 21-day nephritic P-selectin–null mice (lanes 3 and 5, respectively).

A: effectiveness of relevant Ca_v3 depletion was confirmed by Western blot analysis. Cells transfected with Ca_v3.1 small-interfering RNA (siRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA were compared with untreated jugular vein endothelial cells (VEC) and tracheal venular endothelial cells (TvEC). Immunoblots were compared within the same membrane, and actin served as a loading control. B: P-selectin expression after 20% elongation and after transfection with Ca_v3.1 siRNA. VEC transfected with Ca_v3.1 siRNA did not increase P-selectin expression, yet control transfected cells (20% + GAPDH) showed the expected increase in P-selectin expression after cyclic stretch (mean ± SE, n = 4 experiments; *P < 0.05 vs. medium and GAPDH). C: P-selectin expression was confirmed by enzyme-linked immunosorbent assay (ELISA) after 20% elongation or thrombin and after transfection with Ca_v3.1 siRNA. VEC transfected with Ca_v3.1 siRNA did not increase P-selectin expression, yet control transfected cells (20% + GAPDH) and medium controls showed the expected increase in P-selectin expression after cyclic stretch. Likewise, thrombin-induced P-selectin expression was similar to control cells transfected with GAPDH siRNA. However, cells transfected with Ca_v3.1 siRNA showed a significant reduction in P-selectin expression after thrombin. Both groups treated with Ca_v3.1 siRNA were statistically different from medium and GAPDH groups (mean ± SE, n = 3 experiments/group; ***P = 0.001 vs. medium and GAPDH). D: P-selectin expression after 20% elongation and after transfection with Ca_v3.1 siRNA. TvEC transfected with Ca_v3.1 siRNA did not increase P-selectin expression, yet control transfected cells (20% + GAPDH) showed the expected increase in P-selectin expression after cyclic stretch (mean ± SE, n = 4 experiments; **P = 0.01 vs. medium and GAPDH).