fibrin and glycyl-arginyl-glycyl-aspartyl-serine

alpha-Thrombin stimulation of human platelets initiates inside-out signaling to integrin alpha(IIb)beta(3) (glycoprotein IIb/IIIa), resulting in the exposure of ligand binding sites. In the present study, the regulation of alpha(IIb)beta(3) via protein kinases was investigated in platelets permeabilized with streptolysin O by introducing peptides that interfere with these enzymes and with possible regulatory domains in the cytosolic tail of the beta(3) subunit. Compared with intact platelets, the permeabilized platelets preserved >80% of the aggregation, secretion, and alpha(IIb)beta(3) ligand binding capacity. The peptide YIYGSFK, a substrate for Src kinases, inhibited alpha-thrombin-induced ligand binding to alpha(IIb)beta(3), but a reversed peptide with Y-->F substitutions (KFSGFIF) had no effect. Ligand binding to alpha(IIb)beta(3) was also inhibited by the peptide RKRCLRRL, which binds irreversibly to the catalytic domain of protein kinase C. Peptides corresponding to parts of the protein C inhibitor and beta(2)-glycoprotein I were used as negative controls and failed to interfere with ligand binding. Possible target domains for protein kinases are present in the cytoplasmic tail of the beta(3) subunit. The LLITIHDR peptide, matching the membrane-proximal domain of beta(3) (residues 717 to 724), had no effect, but NNPLYKEA (residues 743 to 750), EATSTFTN (residues 749 to 756), and TNITYRGT (residues 755 to 762), which mimicked overlapping domains of the carboxy-terminal part of beta(3), reduced alpha-thrombin-induced ligand binding by 60+/-4%, 97+/-1%, and 97+/-2% (n=3) at 500 micromol/L peptide, respectively. These observations indicate that Src kinases and protein kinase C take part in inside-out signaling to integrin alpha(IIb)beta(3) and identify target domains in beta(3) that contribute to the regulation of this integrin.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Blood Platelets; Cell Membrane Permeability; Enzyme Inhibitors; Fibrin; Fibrinogen; Fibronectins; Humans; Oligopeptides; Peptides; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Protein Kinase Inhibitors; src-Family Kinases; Streptolysins; Thrombin

We investigated the adhesion of human umbilical vein endothelial cells (HUVECs) to fibrin(ogen) molecule of varying structure for identifying sites that mediate cell attachment. Fibrin was prepared either with ancrod which liberates only FPA from fibrinogen, or with thrombin, which liberates both FPA and FPB. Both fibrin preparations equally supported HUVEC attachment. GRGDS, RGD-containing peptides of snake venoms, and monoclonal antibodies against alpha v beta 3 (23C6 and 7E3) inhibited the attachment of HUVECs to fibrin by 65-75%. In contrast, the attachment of HUVECs to fibrinogen was less effective and was almost completely inhibited by both RGD-containing peptides and by antibodies against integrin alpha v beta 3 (85-95% inhibition). The C-terminal dodecapeptide of fibrinogen gamma chain (residues 400-411) inhibited minimally the attachment of HUVECs to fibrin. Additionally, the binding of RGD-containing snake venom peptides to HUVECs was both RGD- and divalent-cation-dependent. The IC50S for inhibition of HUVEC attachment to fibrin were 0.09 microM (rhodostomin), 1.54 microM (trigramin) and 1.64 microM (halysin). These results indicate that fibrin mediated support of cell attachment is independent of the cleavage of FPB from fibrinogen. HUVEC attachment to fibrinogen was almost completely inhibited by RGD-containing peptides and by antibodies against alpha v beta 3. In contrast, the attachment to fibrin was partially resistant to RGD-containing peptides and to the monoclonal antibodies against integrin alpha v beta 3. However, alpha v beta 3 is the major receptor mediating HUVEC attachment to fibrin.

Topics: Amino Acid Sequence; Cell Adhesion; Cells, Cultured; Crotalid Venoms; Endothelium, Vascular; Fibrin; Fibrinogen; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptides; Receptors, Vitronectin; Snake Venoms; Umbilical Veins

We have investigated the adhesion and spreading of platelets on polymerized fibrin of varying structure to identify sites that mediate these interactions. Fibrin was prepared with thrombin to remove both fibrinopeptide A (FPA) and fibrinopeptide B (FPB) and with reptilase or Agkistrodon contortrix thrombin-like enzyme (ACTE) to selectively remove FPA or FPB, respectively. Residual fibrin-bound enzymes were inhibited with D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK). Platelet adhesion was independent of fibrinopeptide cleavage and was equal on fibrin prepared with each of the three enzymes. In contrast, FPB cleavage increased spreading as quantitated by fluorescence microscopy of platelets stained for glycoprotein IIb-IIIa. The 24% +/- 4% spreading on reptilase-fibrin was significantly less than the 70% +/- 8% on thrombin-fibrin or 65% +/- 9% on ACTE-fibrin (P < .0005 for both). Protease III from Crotalus atrox venom was used to specifically cleave residues B beta 1-42 from fibrinogen to further investigate the role of the beta chain N-terminus in promoting platelet spreading. After clotting with thrombin, this fibrin derivative lacked beta 15-42 and supported significantly less spreading. A monoclonal antibody (MoAb) reactive with beta 15-21 inhibited spreading on thrombin-fibrin as did peptide beta 15-42, while control MoAbs and peptides had no significant effect. These results indicate that adhesion and spreading of platelets on fibrin are mediated by different interactions, and that spreading can be mediated by FPB cleavage and the amino terminus of the beta chain including residues beta 15-42.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Antibodies, Monoclonal; Blood Platelets; Cell Movement; Electrophoresis, Polyacrylamide Gel; Fibrin; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; In Vitro Techniques; Molecular Sequence Data; Oligopeptides; Platelet Adhesiveness; Serotonin

We have previously reported that fibrinogen/fibrin can induce the migration of vascular smooth muscle cells in vitro. In this study, we examined the effect of substrate-bound fibrinogen/fibrin and other cell attachment-promoting proteins on the adhesion of vascular smooth muscle cells. The amount of fibrinogen/fibrin adsorbed to plastic wells and the adhesion of smooth muscle cells to the wells were found to depend on the concentration of fibrinogen used for coating the wells. The effect of fibrinogen/fibrin was comparable to that of so-called cell attachment-promoting proteins (fibronectin, vitronectin, and type I collagen). Adhesion of smooth muscle cells to fibrinogen/fibrin-coated wells was inhibited by the synthetic peptide GRGDS, but not by a control peptide, GRGES. Vitronectin, fibronectin, type I collagen, denatured type I collagen and commercial gelatin also induced smooth muscle cell adhesion. The adhesion induced by vitronectin, denatured type I collagen, and commercial gelatin was inhibited by GRGDS. However, the adhesion induced by type I collagen was not influenced and that induced by fibronectin was only slightly inhibited. These observations suggest that fibrinogen/fibrin deposited extracellularly in the arterial intima may act as a scaffold in the process of smooth muscle cell migration.

Topics: Animals; Cattle; Cell Adhesion; Cells, Cultured; Collagen; Fibrin; Fibrinogen; Fibronectins; Gelatin; Glycoproteins; Muscle, Smooth, Vascular; Oligopeptides; Vitronectin