Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen.
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.
Barnes
C S
CS
Department of Cardiovascular Research, Merck KGaA, Frankfurterstrasse 250, 64271 Darmstadt, Germany. christopher.barnes@merck.de
Krafft
B
B
Frech
M
M
Hofmann
U R
UR
Papendieck
A
A
Dahlems
U
U
Gellissen
G
G
Hoylaerts
M F
MF
D000818:Animals; D003094:Collagen / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007865:Leeches / Q000737:chemistry; D010843:Pichia / Q000235:genetics; D010973:Platelet Adhesiveness / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D012471:Salivary Proteins and Peptides / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism
D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C440095:saratin; D014841:von Willebrand Factor; D003094:Collagen