Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule

• Published in: Blood Vol. 93; no. 3; pp. 876 - 885
• Main Authors: Evangelista, Virgilio, Manarini, Stefano, Sideri, Rita, Rotondo, Serenella, Martelli, Nicola, Piccoli, Antonio, Totani, Licia, Piccardoni, Paola, Vestweber, Dietmar, de Gaetano, Giovanni, Cerletti, Chiara
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01.02.1999; The Americain Society of Hematology
• Subjects: Adult; Animals; Biological and medical sciences; Blood Platelets - metabolism; Calcium - physiology; CD18 Antigens - physiology; Cell Adhesion; Cell interactions, adhesion; CHO Cells; Cricetinae; Cricetulus; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Genistein - pharmacology; Humans; Macrophage-1 Antigen - physiology; Magnesium - physiology; Membrane Glycoproteins - antagonists & inhibitors; Membrane Glycoproteins - physiology; Molecular and cellular biology; Neutrophils - physiology; P-Selectin - genetics; P-Selectin - physiology; Phosphorylation - drug effects; Platelet Activation; Platelet Adhesiveness - physiology; Protein Processing, Post-Translational - drug effects; Protein-Tyrosine Kinases - antagonists & inhibitors; Protein-Tyrosine Kinases - physiology; Recombinant Fusion Proteins - physiology; Transfection; Human; Endothelial cell; Diapedesis; Leukocyte; Pathogenesis; Granulocyte; Cardiovascular disease; Cell cell interaction; Inflammation; Adhesion; Thrombosis; Vascular disease; P Selectin; Platelet; Integrin
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V93.3.876

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.