title: von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion.
abstract: Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.
authors.affiliation: Departments of Chemical and Biological Engineering.
authors.forename: Sri R
authors.initials: SR
authors.lastname: Madabhushi
authors.forename: Chengwei
authors.initials: C
authors.lastname: Shang
authors.forename: Kannayakanahalli M
authors.initials: KM
authors.lastname: Dayananda
authors.forename: Kate
authors.initials: K
authors.lastname: Rittenhouse-Olson
authors.forename: Mary
authors.initials: M
authors.lastname: Murphy
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Ryan
authors.forename: Robert R
authors.initials: RR
authors.lastname: Montgomery
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D000328:Adult; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects / Q000235:genetics; D010973:Platelet Adhesiveness / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D011498:Protein Precursors; D014841:von Willebrand Factor