The structure of the A3 domain suggests that adhesion to collagen is primarily achieved through interactions between negatively charged residues on A3 and positively charged residues on collagen. The absence of a pronounced binding groove precludes a large van der Waals surface interaction between A3 and collagen and is consistent with the low affinity for collagen of a single A3 domain and the requirement for multimeric vWF for tight association with collagen. The absence of bound metal ions upon soaking the crystal in MgCl2 and vWF A3's conformational incompatibility for metal binding is consistent with the absence of a functional role for metal ion binding in A3, which contrasts the metal ion activation required for ligand binding by the homologous integrin I type domains.
lntroduction a more gradual drop at higher shear rates above 2000/sec. Aggregates were larger in the single pass system. Measurements of B-thromboglobulin in the outflow of the chamber showed a slight but significant increase in the recirculating system indicating that more platelet activation occurred in this system. The difference between the single pass and the reperfusion system was not observed for collagen type I and III. The role of divalent cations in adhesion to collagen type IV was compared to that of adhesion to collagens type I and III and to endothelial cell marix. Adhesion at 1600/sec was very sensitive to the Mg'* concentration for collagen type IV whereas little effect was seen at 300/sec. Also platelet adhesion to endothelial cell matrix and to collagen type III were sensitive to the Mg'* concentration at 1600/sec but the effect was less than for collagen type IV. Adhesion to collagen type I was not very sensitive to Mg2*. Aggregate formation on collagen type IV and I was strongly impaired at low Mg'*, but aggregates on collagen type III were almost not affected. These results indicate that variations in the physiological range of Mg2* may have an effect on platelet adhesion in vivo. The Mg'* concentration may have an effect on bleeding from wounds. Platelet adhesion to collagen type VI was much stronger at 100/sec (60 Vo coverage) than at 1000/sec (6Vo) (6). In contrast to the study by Saelman, however, aggregation at 100/sec was stronger for collagen type VI than for collagen type I (4).This may be due to differences in the collagen type VI, that was used. Ross et al used collagen type VI purified from umbilical cords in which the globular domains were intact. Another difference was the use of a single pass system instead of the recirculating system of Baumgartner e[ al. that was used in the earlier study. Adhesion to collagen type VI required von Willebrand Factor (vWF) and was inhibited by antibodies to GPIb and by aurin tricarboxylic acid. More unexpected was the observation that an antibody to GPIIb-IIIa was inhibitory not only for platelet aggregation but also almost completely abolished adhesion to collagen type VI whereas less effect was seen on adhesion to collagen type I. It is important to note that these studies were performed in citrated blood. The effect of GPIIb-IIIa inhibition on adhesion to collagen type I is strong in citrated blood and almost absent in heparinized blood. Collagen types Platelet adhesion to collagen is an essential first step in haemostasis and thrombosis. After platelets have adhered they become activated, other platelets stick to the adhering platelets and a haemostatic plug or platelet thrombus is formed.
Thrombotic thrombocytopenic purpura (TTP) after bone marrow transplantation (BMT) differs from classic TTP in its clinical course and therapy. A characteristic of classic TTP is the inhibition of a plasma protease that specifically cleaves von Willebrand factor (vWF), thus reducing its multimeric size. We investigated whether this protease was also inhibited in BMT-associated TTP. Plasma from patients with classic or BMT-associated TTP was incubated with recombinant vWF R834Q, a vWF mutant with enhanced sensitivity to the protease. The proteolysis of vWF multimers was analyzed and quantified on Western blot. Metalloprotease activity was strongly inhibited in the classic TTP patient group. However, metalloprotease activity was normal in the BMT-associated TTP patient group. The difference in activity between the two patient groups was highly significant (P = .0016). The results indicate that the etiologies of classic and BMT-associated TTP are indeed different and provide an explanation for the lack of success of plasma exchange in BMT-associated TTP.
SummaryBinding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the threedimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997; 5: 1147). We hypothesized that the top face of this domain might be the collagen-binding site. Based on this hypothesis, we made seven vWF mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, and R1074A). Collagen binding of these mutants was investigated in ELISA and with Surface Plasmon Resonance (BIAcore). In addition, we studied collagen binding of mutants lacking the A2 or D4 domains, which flank the A3 domain.In ELISA, all point mutants and deletion mutants bound to collagen in amounts similar to wild type (WT)-vWF. In the BIAcore we found that WT-vWF has an apparent KD for collagen of 1-7 nM on a subunit base. The apparent kinetic parameters of the point mutants and deletion mutants were not significantly different from WT-vWF, except for DA2-vWF, which had a lower KD, indicating that the A2 domain somehow modulates binding of vWF to collagen type III.Based on our results, we conclude that the amino acid residues mutated by us are not critically involved in the interaction between vWF and collagen type III, which suggests that the collagen binding site is not located on the top face of the A3 domain.
Molecular modeling techniques have been used to derive a substrate model for class mu rat glutathione S-transferase 4-4 (GST 4-4). Information on regio- and stereoselective product formation of 20 substrates covering three chemically and structurally different classes was used to construct a substrate model containing three interaction sites responsible for Lewis acid--Lewis base interactions (IS1, IS2, and IS3), as well as a region responsible for aromatic interactions (IS4). Experimental data suggest that the first protein interaction site (pIS1, interacting with IS1) corresponds with Tyr115, while the other protein interaction sites (pIS2 and pIS3) probably correspond with other Lewis acidic amino acids. All substrates exhibited positive molecular electrostatic potentials (MEPs) near the site of conjugation with glutathione (GSH), as well as negative MEP values near the position of groups with Lewis base properties (IS1, IS2, or IS3), which interact with pIS1, pIS2, or pIS3, respectively. Obviously, complementarity between the MEPs of substrates and protein in specific regions is important. The substrate specificity and stereoselectivity of GST 4-4 are most likely determined by pIS1 and the distance between the site of GSH attack and Lewis base atoms in the substrates which interact with either pIS2, pIS3, or a combination of these sites. Interaction between aromatic regions in the substrate with aromatic amino acids in the protein further stabilizes the substrate in the active site. The predictive value of the model has been evaluated by rationalizing the conjugation to GSH of 11 substrates of GST 4-4 (representing 3 classes of compounds) which were not used to construct the model. All known metabolites of these substrates are explained with the model. As the computer-aided predictions appear to correlate well with experimental results, the presented substrate model may be useful to identify new potential GST 4-4 substrates.
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