Abstract
Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.
Highlights
Ca2+(Kc. = 0.4 PM) but is not dependent on platelet secretion
Platelet-specific monoclonal antibodies were generated by fusing the murine myeloma cell line, SP-2, with splenic lymphocytes from a mouse that had been immunized with platelets from a patient with Glanzmann's thrombasthenia.These platelets contained approximately 5% of the normal amount of glycoproteins IIb and IIIa as measured by the binding of IIb- and IIIa-specific monoclonal antibodies.Platelet-specific clones were screened further for preferential reactivity with
PAC-1 Binding to Platelets-Antibody binding studies were performed with radioiodinated PAC-1. ’251-PAC-b1ound minimally to eitherunstimulated gel-filtered platelets or toplatelets incubated with 1p~ PGI, to prevent platelet activation
Summary
The final substrate in the assay was orthophenylenediamine, and the reaction product was quantitated at 405 nm with a Multiscan spectrophotometer [18]. 1was shown to be an IgM K using a mouse immunoglobulin subtype identification kit (Boehringer Mannheim) It was purified from 15 to 20 mlof ascites by precipitating the protein in 20 volumes of 2% boric acid for 30 min at room temperature, redissolvingthe precipitate in 5-10 ml of PBS, and applying the sample to a 1X 97 cm Sepharose. The platelet proteins were transferred from the gel to positively charged Zeta-bind paper (American Cyanamid) [23], and protein bands reacting with PAC-1 were identified using peroxidase-conjugated goat anti-mouse immunoglobulin [20]. After incubation of the platelets with the antibody for 15 min, platelets and bound antibody were separated from free antibody by filtration on 0.45-pm nitrocellulose filters that had been presoaked in 6% albumin. After 5 min, aggregation was initiated by stirring the platelets in an aggregometerat 37 "C and adding 200 pg/ml fibrinogen and 100 p~ CaC12.The extent of aggregation and ['4C]serotonin release 3 min later was measured [19]
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