Measurement of the binding forces between von Willebrand factor and variants of platelet glycoprotein Ibalpha using optical tweezers.
OBJECTIVE
Thrombus formation is initiated by adhesion of the platelet receptor, glycoprotein (GP) Ib-IX-V complex, to its adhesive ligand, von Willebrand factor (vWf), in the subendothelium or plasma. The vWf-binding domain of GP Ib-IX-V is in the GP Ibalpha subunit of the complex and contains a leucine-rich repeat region. The adhesion of different leucine-rich repeats was studied using optical tweezers in order to determine which ones were critical for the vWf/GP Ibalpha interaction.


METHODS
Canine GP Ibalpha does not normally bind to human vWf, and thus canine-human GP Ibalpha chimeras were constructed by sequentially replacing human GP Ibalpha structural regions with their canine counterparts. Chinese hamster ovary (CHO) cells, which are frequently used to express platelet GP complexes, were transfected with the chimeric proteins. Optical tweezers (lambda = 830 nm) were used to investigate bond strengths between vWf and different GP Ibalpha canine-human chimeras. Since vWf does not bind GP Ibalpha without high shear stress, the compounds botrocetin and ristocetin were used to induce binding between human vWf and the chimeras.


RESULTS
All human-canine GP Ibalpha chimeras bound to vWf in the presence of botrocetin. Replacement of the N-terminal flanking sequence and the first leucine-rich repeat resulted in lower GP Ibalpha/vWf bond strengths than the wild-type human GP Ibalpha/vWf bond strength (P < 0.05). Chimeras lacking the second leucine-rich repeat did not adhere to vWf with ristocetin acting as modulator.


CONCLUSIONS
The N-terminal flanking sequence and the first leucine-rich repeat of GP Ibalpha were found to be important but not necessary for GP Ibalpha to adhere to vWf. The second leucine-rich repeat was found to be critical for GP Ibalpha to bind vWf and could potentially be used in the development of a novel recombinant anti-thrombotic drugs.
Arya
Maneesh
M
Department of Bioengineering, Rice University, Houston, Texas 77005, USA.
López
José A
JA
Romo
Gabriel M
GM
Dong
Jing-Fei
JF
McIntire
Larry V
LV
Moake
Joel L
JL
Anvari
Bahman
B
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003435:Crotalid Venoms / Q000494:pharmacology; D004285:Dogs; D006487:Hemostasis / Q000502:physiology; D006801:Humans; D007834:Lasers; D007930:Leucine; D010973:Platelet Adhesiveness; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins; D012310:Ristocetin / Q000494:pharmacology; D013927:Thrombosis / Q000503:physiopathology; D014841:von Willebrand Factor / Q000502:physiology*
D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin; D007930:Leucine