Abstract
We have used recombinant von Willebrand factor (vWF) fragments to investigate the properties regulating A1 domain interaction with platelet glycoprotein (GP) Ibalpha. One fragment, rvWF508-704, represented the main portion of domain A1 (mature subunit residues 497-716) within the Cys509-Cys695 disulfide loop. The other, rvWF445-733, included the carboxyl-terminal region of domain D3, preceding A1, and corresponded to the proteolytic fragment originally identified as the GP Ibalpha-binding site (residues 449-728). Conformational changes were induced by reduction and alkylation of the Cys509-Cys695 bond and/or exposure to acidic pH. The cyclic rvWF445-733 fragment exhibited the function of native vWF A1 domain. When immobilized onto a surface, it tethered platelets at shear rates up to 6,300 s-1 mediating low velocity translocation but not stable attachment; in solution, it exhibited limited interaction with GP Ibalpha. In contrast, fragments with perturbed conformation could not tether platelets at high shear rates but promoted stable adhesion at lower shear and bound tightly to GP Ibalpha. Only in the presence of the exogenous modulator, botrocetin, did cyclic rvWF445-733 mediate irreversible adhesion. Thus, conformational transitions in the vWF A1 domain may influence differentially the efficiency of bond formation with GP Ibalpha and the stability of binding.
Highlights
The A1 domain of von Willebrand factor1 immobilized onto exposed surfaces at sites of vascular injury initiates platelet adhesion and thrombus formation by interacting with the glycoprotein (GP) Ib␣ receptor [1,2,3]
Coating of von Willebrand factor (vWF) A1 Domain Fragments onto a Glass Surface—Preliminary experiments established that platelet adhesion to immobilized native vWF or recombinant fragments was maximal after coating glass with a solution at the concentration of 100 g/ml for 1 h at room temperature (22–25 °C)
It is apparent that conformational changes within the A1 domain of vWF can enhance its capacity to block GP Ib␣ in solution but at the expense of a decrease in the normal function of tethering platelets to a surface under high flow conditions. These results suggest that structural modifications reducing the dissociation rate of the interaction between vWF A1 domain and GP Ib␣, a feature required for good inhibitory function in solution, may result in a slower association rate with consequent decreased efficiency in initiating platelet adhesion
Summary
Preparation of Reconstituted Blood—Blood was collected from healthy and medication-free donors into polypropylene syringes containing as anticoagulant the ␣-thrombin inhibitor D-phenylalanyl-Lprolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK) at the final concentration of 50 M. Platelet-depleted reconstituted blood was prepared by centrifuging the final cell suspension at 150 ϫ g for 15 min, removing the resulting platelet-rich supernatant fluid, and replacing it with an equivalent volume of Hepes/Tyrode buffer, pH 7.4, containing 50 mg/ml bovine serum albumin. The inhibitory effect of specific monoclonal antibodies on platelet interaction with immobilized native vWF or recombinant fragments was assessed by incubating purified IgG with reconstituted blood at room temperature for 20 min before perfusion through the chamber. Platelet-rich plasma was prepared by centrifugation of blood containing PPACK at 150 ϫ g for 15 min at room temperature and diluted in 10 mM Hepes buffer, pH 7.4, to give a final platelet count of 1 ϫ 108/ml. The amount of protein adsorbed onto the surface of glass coverslips was determined by subtracting from the total initial amount in the coating solution the sum of that recovered in the supernatant solution after coating and in all washing solutions
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