Measurement of Glycoprotein llb/llla Blockade by Flow Cytometry with Fluorescein Isothiocyanate-conjugated Crotavirin, a Member of Disintegrins

Abstract

SummaryThe blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab’)2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin’s binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein Ilb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein Ilb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein Ilb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin’s binding at the saturation dose reflects the extent of glycoprotein Ilb/IIIa blockade by 7E3. At the saturation binding concentration (5 |xg/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein Ilb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein Ilb/IIIa of platelets in the 7E3 or 7E3 F(ab’)2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 fig/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein Ilb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Antithrombotic Effect of a Protein-Type I Class Snake Venom Metalloproteinase, Kistomin, Is Mediated by Affecting Glycoprotein Ib-von Willebrand Factor Interaction

“…Citrated blood was immediately centrifuged for 10 min at 120g and 25°C, and the supernatant [platelet-rich plasma (PRP)] was obtained. Human washed platelet suspension (PS) was prepared as described previously (Liu et al, 1996) and adjusted to approximately 3.8 ϫ 10 8 platelets/ml. Platelet aggregation was monitored by light transmission in a Lumi-Aggregometer (Chrono-Log, Havertown, PA) with continuous stirring at 900 rpm at 37°C as described previously (Liu et al, 1996).…”

Aggretin, a snake venom–derived endothelial integrin α2β1 agonist, induces angiogenesis via expression of vascular endothelial growth factor

“…To study the target site of aggretin on endothelial cells, we first conjugated aggretin and BSA with FITC by a previously described method, 43 and examined their binding reactions toward HUVECs by flow cytometry. After incubation of FITC-conjugated aggretin with HUVECs in the presence of Ca 2ϩ (5 mM), the increment of relative fluorescence intensity of the bound FITC-aggretin was concentration dependent and reached a saturated binding at a concentration of 10 M ( Figure 3A).…”

Rhodostomin, a Disintegrin, Inhibits Adhesion of Neutrophils to Fibrinogen and Attenuates Superoxide Production

“…Rhodostomin and bovine serum albumin (BSA) were conjugated with FITC following the protocol described by Liu et al [19]. The fluorescence intensities of FITC-conjugated rhodostomin and BSA were measured using a CytoFluor 2300 fluorescent plate reader (Millipore).…”

“…Owing to the overlapping binding site, 7E3 has been used experimentally as a binding competitor in displacing disintegrin binding to the platelet · IIb ß 3 [19] or endothelial · V ß 3 integrins [16]. 7E3 interacts with leukocytes via Mac-1; however, the functional role of disintegrins on leukocytes has not been established.…”