Abstract
We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.
Highlights
We demonstrate that unstimulated platelets attach t(oalso designatedal&) is a well-characterized adhesion recepimmobilized fibrinogenin a selective process mediated tor essential for platelet aggregation [1, 2] and is involved in by the membrane glycoprotein (GP) complex IIb-IIIah. e initial attachment, independenotf platelet activation, is followed by spreading and irreversible adhesion evenin thepresence of activation inhibitors
G P IIb-IIIa on activated platelets serves as a receptor for fibrinogen,fibronectin ( l l ), vitronectin (12, activated platelets adhere to immobilized fibrinogen 13)and von Willebrand factor [14]
GPIIb-IIIahas been llW peilmbailrtilaeteenlbeddrtascnfaafdpacfiataloccrittt.ooyrTatthdohehrsoaeeudreghrheetroseuGvlttiosPtrIfodIinebbmer-oIcoItnIineansc,ttuariannntdsetaihnmvthadouvanlteaWtGoeindPlal-yIIbpiimtr-sopstlepiicencasitfehidcasacsanptohatecbisteyuerntfoacclieneatrerelrycaecdptetfwoinrietihdn.vsouWlrvfeeadcreeia-nbsootnhueinsddftuahdnahctetistoihvnies, IIIa on unstimulated platelets displays a recognition is a crucial point for understanding the potentialrole of G P specificity for attachment to immobilized adhesive pIrIob--IIIa in the initiation of thrombogenesis.we teins that is distinct from that seen following platelet have undertaken the present seorfieesxperiments to compare activation
Summary
Fragments X and D gave essentially identical results, confirming that the AaIIR-!IH Arg-Gly-Asp-Phe sequence, present in Activation-independentPlateletAdhesiontoImmobilized Fibrinogen “Unstimulated platelets readily adhered to immobilized fibrinogen, but not tobovine serum albumin (BSA; Fig. 1).Prior treatmentof platelets with the activation inhibitors, theophylline(0.3mM), forskolin (3.3 p ~ )pr, ostaglandin E, (PGE,; 2 p ~ ) a,nd with the ADP scavenger, apyrase (5 units/ml), did notimpairtheiradhesioncapacitytoward fragment X but not in fragment D (see Miniprint), has no detectable influence in supporting adhesion of nonactivated platelets, as alsosuggested by the negative findings obtained withfragment E (Fig. 4). Unstimulatedplatelets failed to adhere to short synthetic peptides(13-16 residues in length) reproducing the two Arg-Gly-Asp sequences in the Aa chain immobilized fibrinogen, itcompletely inhibited their orthecarboxylterminus of the y chain(seeMiniprint); ability to binsdoluble fibrinogen after treatment with units/ ml a-thrombin (Fig. 1).Scanning electron microscopy demmoreover, monoclonalantibodiesreacting with these peptides and the corresponding sequences in the intact onstrated nonactivated platelet morphology before addition fibrinogen molecule (see Miniprint)failed to inhibit adhesion to immobilized fibrinogen,but extensivepseudopod formation of unstimulated platelets to surface-bounfdibrinogen (results and spreading after attachment to fibrinogen-coated surfaces, despite the presenceof platelet activation inhibitors (Fig. 2). Platelets were detected using “‘1-labeled monoclonal antibodyLJ-P4,as described under“ExperimentalProcedures.’’ Adhesion of stimulated platelets,0;adhesion of unstimulated sion of unstimulated platelets to fibrinogen and the fibrino- platelets, 0; adhesion of unstimulated platelets in the presence of gen-derived fragments (Fig. 4) This antibody inhibited monoclonal antibody LJ-CP3 (50 pg/ml), 0. 0.5 h tin, phenymethylsulfonyl fluoride, aprotinin, or hirudin, indicating that plasmin or thrombin, possibly present as trace a b cedbecadbecadbecedbecadbecadbec d e
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