Production and Characterization of Saratin, an Inhibitor of von Willebrand Factor-Dependent Platelet Adhesion to Collagen

ABSTRACT

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 (IC₅₀ = 0.23 ± 0.004 and 0.81 ± 0.04 μg mL^-1, respectively) and to calf skin collagen (IC₅₀ = 0.44 ± 0.008 μg 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 (IC₅₀ = 0.96 ± 0.25 μg mL^-1), but at reduced shear (1300 s^-1) a rightward shift in the dose-response curve was noted (IC₅₀ = 5.2 ± 1.4 μg mL^-1). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K_d 5 × 10^-8 M and 2 × 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.

KEYWORD

Saratin - von Willebrand factor - platelet adhesion - antithrombotic agent - Hansenula polymorpha

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