title: Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses.
abstract: With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2-loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i, followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, "balloon"-like structures (diameter about 5 microm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i, by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.
authors.affiliation: Department of Human Biology, University of Maastricht, The Netherlands.
authors.forename: J W
authors.initials: JW
authors.lastname: Heemskerk
authors.forename: W M
authors.initials: WM
authors.lastname: Vuist
authors.forename: M A
authors.initials: MA
authors.lastname: Feijge
authors.forename: C P
authors.initials: CP
authors.lastname: Reutelingsperger
authors.forename: T
authors.initials: T
authors.lastname: Lindhout
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects* / Q000648:ultrastructure; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects* / Q000648:ultrastructure; D002614:Chelating Agents / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005340:Fibrinogen / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008563:Membrane Lipids / Q000097:blood / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D018715:Microscopy, Video; D019007:P-Selectin / Q000096:biosynthesis; D010718:Phosphatidylserines / Q000097:blood / Q000502:physiology*; D010766:Phosphorylation; D010973:Platelet Adhesiveness / Q000187:drug effects* / Q000502:physiology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011760:Pyrrolidinones / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D004963:Estrenes; D008563:Membrane Lipids; D019007:P-Selectin; D010718:Phosphatidylserines; D010975:Platelet Aggregation Inhibitors; D011760:Pyrrolidinones; C074854:U 73343; D000001:Calcimycin; D004533:Egtazic Acid; D005340:Fibrinogen; D003094:Collagen; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D013917:Thrombin; D001241:Aspirin; D002118:Calcium; D000077191:Wortmannin