Antithrombotic map of the N-terminal domain of the GPIIIa subunit of the human platelet fibrinogen receptor (GPIIb-IIIa) determined in vivo by monoclonal, immunochemical inhibition of acute arterial thrombosis.
The inhibition of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa (GPIIb-IIIa) or integrin alphaIIbbeta3, has recently became an accepted practice in clinical cardiology. The interest lies now in the improvement of the antithrombotic activity and the minimization of the secondary effects of the receptor inhibitors, by their evaluation in vivo in the different dynamic conditions and pathological states under which these inhibitors have to perform. In this paper, we functionally map in vivo the N-terminal domain of the GPIIIa subunit, using the antithrombotic activity of five murine monoclonal antibodies (mabs) (P37, P40, 95-1, P95-2 and P97), all of them inhibitors of platelet aggregation in vitro and directed to this ligand binding domain of the human fibrinogen receptor. Competition experiments have shown that these mabs bind with high affinity (5-7 nM) and compete very strongly among themselves for binding to human resting platelets, except P40, which neither binds nor competes. These antibodies were assayed in a dog model of acute thrombosis in the carotid artery, which were induced 15 min after their intravenous administration (0.8 mg/kg). The antithrombotic activity was quantified by the measurement of the [111In]oxine-labelled platelet deposition at the site of the arterial lesion and was expressed as the percentage of the total circulating platelets. Antibody P37, directed to the GPIIIa 101-109 sequence, decreased the platelet deposition 630-fold with respect to control animals. P95-2, P97 and P95-1 decreased the platelet deposition 160-, 32- and 25-fold, respectively, while P40, directed to the GPIIIa 260-302 sequence, did not show any antithrombotic activity. We conclude that all the mabs directed to the N-terminal domain of GPIIIa, which inhibit platelet aggregation in vitro and whose epitopes are very close to each other and exposed in resting platelets, have high antithrombotic activity in vivo, which varies depending on the actual location of the epitopes in the receptor topography. Among these antibodies, P37, the strongest receptor inhibitor in vivo and whose epitope is most probably the closest to the fibrinogen binding site(s), seems the best candidate for comparative studies in animal models with today's best GPIIb-IIIa inhibitors and for clinical trials in humans in order to arrest or prevent thrombosis, reocclusion and late restenosis.
Escudero
C
C
Departamento de Cirugía Experimental, Hospital Puerta de Hierro, San Martín de Porres 4, 28035 Madrid, Spain.
Alvarez
M V
MV
de Haro
J
J
Alvarez
L
L
Millán
I
I
Castillo-Olivares
J L
JL
González-Rodríguez
J
J