RESUMO
Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.
Assuntos
Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Fator de von Willebrand/antagonistas & inibidores , Animais , Colágeno/metabolismo , Colágeno/fisiologia , Humanos , Sanguessugas/química , Pichia/genética , Inibidores da Agregação Plaquetária/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/isolamento & purificação , Fator de von Willebrand/metabolismoRESUMO
This article describes the expression of the hirudin gene heterologously in the methylotrophic yeast Hansenula polymorpha, the establishment of an industrial-scale production process and the subsequent clinical development of polyethylene glycol (PEG)-hirudin. PEGylation increases the molecular weight of hirudin, thereby reducing its kidney filtration rate and immunogenicity and increasing its half-life in the circulation.
