Pac 1

Preconjugated fluorescent antibodies were from BD Biosciences (San Jose, CA, USA) (CD14–phycoerythrin (PE), P-selectin (CD62P)–PE, immunoglobulin G (IgG₁)–PE, CD16–PE-Cy5, CD40L–PE, CD42a–fluorescein isothiocyanate (FITC), PAC-1–FITC, CD147–FITC, TNF-α–FITC, IL-6–FITC, CD61–PerCP, CCR2–Alexa Fluor and BrdU Flow Kit). Anti-CD147 antibody was provided by the Department of Cell Biology, Fourth Military Medical University (Xi’an, China). An anti-IKK beta (IKKβ) antibody was also used (Abcam, Cambridge, UK).

Haematocrit samples of a few of our patients in the cohort were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA) (17). CD62P is a marker for P-selectin that is either found on the membrane of platelets or inside them. PAC-1 identifies platelets through marking the glycoprotein IIb/IIIa (gpIIb/IIIa) on the platelet membrane. To study platelet pathology, 4 µL CD62P and 4 µL PAC-1 was added to 20 µL haematocrit, followed by incubation for 30 min and protected from light at room temperature. The excitation wavelength band for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 to 570 nm and the emission 577 to 607 nm [17 (link)]. Samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).

The whole blood (WB) (haematocrit) samples of healthy volunteers, COVID-19 and Long COVID/PASC patients were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA). CD62P is found in the granules  of platelets and then translocate to the platelet membrane surface. The translocation occurs after the platelet P-selectin is released from the cellular granules during platelet activation [6 (link), 9 (link)]. 4 µL CD62P and PAC-1 was added to 20 µL haematocrit. The haematocrit exposed to the markers was incubated for 30 min (protected from light) at room temperature. The excitation wavelength for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 nm to 570 nm and the emission 577 nm to 607 nm. Processed samples were viewed using the Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).

Wild-type talin-H or talin-H_DM (K15A, R35A) was subcloned into a pEGFP-C1 vector. CHO A5 cells stably expressing integrin α_IIbβ₃ were transfected with empty vector, wild-type talin-H, or talin-H_DM using Lipofectamine 2000 reagent (Life Technologies). After 24 h, cells were stained with anti-α_IIbβ₃ activation-specific mAb PAC1 (1:100 dilution; Catalog# 340535, BD Biosciences) at RT for 40 min, and later incubated with Alexa Flour 647 Goat Anti-Mouse IgM (Catalog# 115-607-020, Jackson Immunology Laboratories; 1:3000 dilution) on ice for 30 min. After wash, cells were fixed and subjected to flow cytometry analysis. Values for PAC1 binding were reported as median fluorescent intensities and the data were normalized to control with empty vector only and are presented by the means ± S.E.M. The two-tailed unpaired t-test was performed to calculate the p value using GraphPad software.

PAC-1 (BD Bioscience) is a ligand-mimetic mAb (IgM) for the activated α_IIbβ₃ integrin (44 (link)). AP3 is a conformation-independent anti-β₃ mAb (45 (link)) and was conjugated with Alexa Fluor 488 (Thermo Fisher Scientific). 10E5 is an anti-α_IIbβ₃ complex specific mAb (31 (link), 46 (link)). 314.5 is an anti-α_IIb mouse mAb that binds to calf-2 domain. H-96 is a rabbit anti-β₃ polyclonal IgG (Santa Cruz Biotechnology). H-160 is a rabbit anti-α_IIb polyclonal IgG (Santa Cruz Biotechnology). PE-labeled MAR4 (BD Bioscience) is a nonfunctional anti-β₁ mAb. PE-labeled TS2/4 (BioLegend) is a nonfunctional anti-α_L mAb (47 (link)). KIM127 that binds to I-EGF-2 domain and mAb 24 (m24) that binds to βI domain are anti-β₂ conformation-dependent mAbs (28 (link), 29 (link), 48 (link), 49 (link)). Rabbit anti-protein C tag was from GenScript. Anti-protein C matrix beads were from Sigma-Aldrich. IRDye 800-labeled streptavidin and IRDye 680-labeled goat anti-rabbit (or mouse) IgG were from LI-COR Biosciences. Human fibrinogen (Plasminogen, von Willebrand Factor, and Fibronectin Depleted) was from Enzyme Research Laboratories. Human fibronectin and human ICAM-1 with a C-terminal Fc tag of human IgG1 (ICAM-1-Fc) were from Sigma-Aldrich and Sino-Biological, respectively.