Marion E. Schiphorst

Platelet Thrombus Formation on Collagen at High Shear Rates Is Mediated by von Willebrand Factor–Glycoprotein Ib Interaction and Inhibited by von Willebrand Factor–Glycoprotein IIb/IIIa Interaction

We studied the role of von Willebrand Factor (vWF) in platelet thrombus formation in flowing blood by using a perfusion system and mutant forms of vWF lacking either interaction with glycoprotein Ib (GpIb) or with glycoprotein IIb/IIIa (alphaIIb-beta3). These mutants were added to the blood of patients with severe von Willebrands disease (vWD) or to normal blood reconstituted with a human albumin solution instead of plasma. This blood was then perfused over collagen type III spray-coated on a glass surface and preincubated for 2 hours with 20 microg/mL plasma vWF. In this way, the adhesion step was mediated by the preincubated plasma vWF bound to collagen type III, whereas thrombus formation was mediated by mutant vWF added to the perfusate. Thrombus formation was absent at all 3 shear rates studied (300, 800, and 2600 s(-1)) when DeltaA1-vWF, lacking interaction with GpIb, was added to the perfusate, indicating the importance of GpIb-vWF interaction for thrombus formation. The interaction of vWF and GpIb is currently thought to be possible under physiological conditions in which the conformation of vWF has been changed by adsorption to a surface. Our results regarding the role of GpIb-vWF interaction in thrombus formation suggest that a second mechanism may operate by which a change may occur in GpIb on the surface of adhered platelets either by activation of the molecule or as a consequence of shear stress. Increased thrombus formation was observed when the Arg-Gly-Gly-Ser-vWF, which does not interact with alphaIIb-beta3, was added to vWD blood and perfused at 2600 s(-1). This increase was not observed in vWD blood at lower shear rates or after addition of Arg-Gly-Gly-Ser-vWF to reconstituted normal blood. Thrombus formation at a high shear rate was largest when either vWF or fibrinogen was present as a single ligand for alphaIIb-beta3 at a high shear rate. When both were present, thrombus formation was decreased. We postulate that thrombus formation is less efficient because of incomplete bridge formation when vWF and fibrinogen are both present as ligands for alphaIIb-beta3.

The effect of ticlopidine administration to humans on the binding of adenosine diphosphate to blood platelets

Administration of Ticlopidine to human volunteers resulted in a prolonged bleeding time and decreased or absent aggregation of platelets with collagen and epinephrine. Adenosine diphosphate (ADP) induced platelet aggregation was initiated by a normal shape change, but the rate of the first wave of aggregation had decreased. The second wave of aggregation was absent. ADP-binding to platelets of volunteers, consisted of two classes of binding sites:one with high affinity and one with low affinity, giving a curvilinear and a linear part in a concentration dependency curve. After Ticlopidine, the low affinity part of the curve had disappeared. Evidence will be presented that this is a specific Ticlopidine effect.

Identification of ectoproteins of human platelets: Combination of radioactive labelling and two-dimensional electrophoresis

Two-dimensional gel electrophoresis combining isoelectroc focussing of reduced or non-reduced proteins in the first dimension with electrophoresis in sodium dodecyl sulfate polyacrylamide gels in the second dimension enabled us to identify 25 ectoproteins in the non-reduced state and 32 in the reduced state. Gel electrophoresis in sodium dodecyl sulfate of non-reduced proteins in the first dimension followed by reduction and gel electrophoresis in sodium dodecyl sulfate in the second dimension was helpful in the identification of the major ectoproteins and indicated that at least seven additional components might be present in the region between 170 and 85 kdaltons. All major ectoproteins could readily be identified. Glycoprotein V showed only a small increase in apparent molecular weight on reduction. It was one of the most basic proteins with a pI value of 6.9. The majority of the ectoproteins were located at an isoelectric point of 5.7 with glycoproteins Ib, IIc, VI and VII as the most acidic components. A good agreement was observed between the thaee labelling techniques which indicates that almost all ectoproteins are glycoproteins containing sialic acid.

Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain

The first step in both normal haemostasis and arterial thrombosis is the interaction between collagen, von Willebrand factor (vWF), and glycoprotein Ib. The A3 domain of vWF forms the principal binding site for collagen type I and type III. Inhibition of the vWF‐collagen interaction by an anti‐human vWF monoclonal antibody (MoAb) 82D6A3 can be a potential way to prevent arterial thrombosis. Identification of the epitope of MoAb 82D6A3 showed recently that the consensus sequence SPWR obtained by phage display could adopt the conformation of the discontinuous epitope. Modelling showed that Trp982 in the vWF had to obtain a more solvent accessible conformation. We performed a detailed fluorescence study of Trp982 in the vWF A3. Using the method described by Hellings et al. (Biophys J 2003;85:1894–1902), we were able to identify two different low‐energy Trp982 rotamers and to link them with their experimentally derived fluorescence lifetimes. Fluorescence anisotropy showed no interconversion in the nanosecond timescale between the two different rotameric states. With these experiments, we gather strong indications for the existence of an exposed rotamer conformation and a rotamer that corresponds to the one observed in the X‐ray structure. These results strongly support the modeling work (Vanhoorelbeke et al., J Biol Chem 2003;278:37815‐37821). Proteins 2004.

Identification of ectoproteins of human platelets: II. Proteins of low molecular weight (10 000–43 000)

Abstract The surface exposed proteins in the range of 43 to 10 kDa were studied with a combination of radiolabelling and two-dimensional electrophoresis. Nineteen proteins were found after reduction, nine of which were linked to other polypeptides in the non-reduced state.

Identification of membrane proteins of human blood platelets with a hydrophobic photolabel

A photoactivable glycolipid probe, 12-(4-azido-2-nitrophenoxy)stearoyl[1-14C]glucosamine, was used to label proteins and lipids of platelet membranes. The proteins were analyzed by two-dimensional high-resolution gelelectrophoresis. The labeling patterns showed that three membrane proteins were labeled which were not previously identified by ectolabeling (Sixma, J.J. and Schiphorst, M.E. (1980) Biochim. Biophys. Acta 603, 70-83). Analysis of the lipid fraction showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were labeled by the probe. The distinct labeling of phosphatidylserine strongly suggests that the probe redistributes between the two halves of the bilayer.