title: Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets.
abstract: Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich-plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear-aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445-733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445-733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.
authors.affiliation: Cox Laboratory for Biomedical Engineering, Rice University, Houston, TX 77251.
authors.forename: B R
authors.initials: BR
authors.lastname: Alevriadou
authors.forename: J L
authors.initials: JL
authors.lastname: Moake
authors.forename: N A
authors.initials: NA
authors.lastname: Turner
authors.forename: Z M
authors.initials: ZM
authors.lastname: Ruggeri
authors.forename: B J
authors.initials: BJ
authors.lastname: Folie
authors.forename: M D
authors.initials: MD
authors.lastname: Phillips
authors.forename: A B
authors.initials: AB
authors.lastname: Schreiber
authors.forename: M E
authors.initials: ME
authors.lastname: Hrinda
authors.forename: L V
authors.initials: LV
authors.lastname: McIntire
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000925:Anticoagulants / Q000494:pharmacology; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology; D010477:Perfusion; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D013314:Stress, Mechanical; D013927:Thrombosis / Q000209:etiology / Q000517:prevention & control*; D014841:von Willebrand Factor / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D001312:Aurintricarboxylic Acid