Fibrinogen receptor binding

From a healthy volunteer 200 ml blood is collected. An aliquot of 8.4 ml blood is mixed with 1.4 ml ACD-buffer in polystyrol tubes and centrifuged at 1000 rpm for 15 min. The resulting platelet-rich plasma (PRP) is collected and an aliquot is taken for platelet counting. Ten milliliters of PRP are mixed with 1 ml ACD-buffer (ACD-PRP, pH 6.5) 5 ml portions of ACD-PRP are transferred to plastic tubes and centrifuged at 1600 rpm for 20 min. The resulting supernatant is decanted, and each pellet is resuspended in 500 pi Tyrode buffer C. An aliquot is taken for platelet counting to calculate the loss of platelets. The platelet suspension is then transferred to the Sepharose-packed column which 

For each concentration, samples are tested in triplicate (test tubes No. 72708, Sarstedt). The total volume of each incubation sample is 500 pi. The concentration of 125I-fibrinogen is constant for all samples (10 pg/500 pi). 

The competition reaction is characterized by one buffer value (bidistilled water) and various concentrations of non-labeled fibrinogen or test compound. 

100 pi non-labeled fibrinogen or test drug (various concentrations, 10 1°-10 3 M) 

Non-specific-binding The non-specific binding of 125I-fibrinogen is defined as the radioligand binding in the presence of 10 5 M of non-labeled fibrinogen. 

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Detection of thrombus is another clinically important goal. Contrast enhancement of vascular thrombosis has been achieved with a microbubble having a small peptide covalently attached to its surface that selectively binds to the GPIIb/llla fibrinogen receptor expressed on the surface of activated platelets that attach to thrombi. 

Mechanism of Action An aggregation inhibitor that inhibits the release of adenosine diphosphate from activated platelets, which prevents fibrinogen from binding to glycoprotein Ilb/IIIa receptors on the surface of activated platelets. TherapeuticEffect Inhibits platelet aggregation and thrombus formation. 

Fibrinogen simultaneously binds to GP llb/llla receptors on two separate platelets, resulting in aggregation. 

Platelet glycoprotein Ilb-IIIa receptor binding is given as fmol 125I-fibrinogen/108 platelets or 125I-fibrinogen molecules bound per platelet. 

Figure 2.16 Like the 5-HT receptor ligands 53 and 54 (Figure 2.15), also lotrafiban 56 (failed in phase III clinical trials) and compound 57 differ only in their amine component but are highly selective for different integrins. Lotrafiban binds preferentially to the fibrinogen receptor (integrin GP b/ma), with a selectivity factor of about 4000, whereas 57 binds preferentially to the vitronectin receptor (integrin aj ), with a selectivity factor of 15 000. Overall, this constitutes a selectivity ratio of more than seven orders of magnitude.

An example of the use of peptidic information comes from the work of Pickett et al. (76) using the known tripeptide RGD (Arg-Gly-Asp) (see Fig. 5.11) motif fibrinogen uses for receptor binding (86). A database of 100,000 compounds, which had been seeded with fibrinogen receptor antagonists covering a wide range of structural classes (see Fig. 5.12), was... 

Shattil SJ, Bud shi A, Scrutton MC. Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the Asence of other excitatory agonists. Blood 1989 73 150-158. 

Parise LV and Phillips DR. Reconstitutiem of the purified platelet fibrinogen receptor fibrinogen binding properties of the glycoprotein llb-llla complex. J biol chem 1985 260 10698-10707. 

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