Tyrosine sulfation of glycoprotein I(b)alpha. Role of electrostatic interactions in von Willebrand factor binding.

Glycoprotein I(b)alpha (GP I(b)alpha), the ligand binding subunit of the platelet glycoprotein Ib-IX-V complex, is sulfated on three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279). This posttranslational modification is known to be critical for von Willebrand factor (vWF) binding; yet it remains unclear whether it provides a specific structure or merely contributes negative charges. To investigate this issue, we constructed cell lines expressing GP I(b)alpha polypeptides with the three tyrosine residues converted to either Glu or Phe and studied the ability of these mutants to bind vWF in the presence of modulators or shear stress. The mutants were expressed normally on the cell surface as GP Ib-IX complexes, with the conformation of the ligand-binding domain preserved, as judged by the binding of conformation-sensitive monoclonal antibodies. In contrast to their normal expression, both mutants were functionally abnormal. Cells expressing the Phe mutant failed to bind vWF in the presence of either ristocetin or botrocetin. These cells adhered to and rolled on immobilized vWF only when their surface receptor density was increased to twice the level that supported adhesion of cells expressing the wild-type receptor and even then only 20% as many rolled and rolled significantly faster than wild-type cells. Cells expressing the Glu mutant, on the other hand, were normal with respect to ristocetin-induced vWF binding and adhesion to immobilized vWF but were markedly defective in botrocetin-induced vWF binding. These results indicate that GP I(b)alpha tyrosine sulfation influences the interaction of this polypeptide with vWF primarily by contributing negative charges under physiological conditions and when the interaction is induced by ristocetin but contributes a specific structure to the botrocetin-induced interaction.

Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions.

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region.

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.

Mutation in the leucine-rich repeat of platelet glycoprotein Ib alpha results in defects in its interaction with immobilized von Willebrand factor under flow.

OBJECTIVE: To characterize effects of the GP Ib alpha mutation (A156V) on its interaction with von Willebrand factor (vWf) under high fluid shear stress. METHODS: The residue A156 of GP Ib alpha was converted to a valine and the mutant expressed in CHO cells expressing wild-type GP Ib beta and GPIX. The transfected cells were tested for their interaction with a panel of GP Ib alpha antibodies and for rolling on immobilized vWf under high shear. RESULTS: The mutation led to surface expression of a GP Ib alpha polypeptide that adopted a different conformation at its N-terminus because binding of the GP Ib alpha antibody AN51, which has a binding epitope in the N-terminal 35 residues, was eliminated, whereas binding of the others (AK2, MB45, and SZ2, all of which bind to regions C-terminal to the AN51 epitope) was normal. Mutant-expressing cells could adhere and roll on immobilized vWf under high fluid shear stress and rolled significantly faster than wild-type cells. CONCLUSION: These studies demonstrate that the mutation A156V results in a conformation change at the N-terminus of GP Ib alpha, which leads to an increase in the dissociation rate of the bond between the GP Ib alpha mutant and vWf.

[A mutation of platelet glycoprotein I balpha results in defects in its interaction with immobilized vWF].

OBJECTIVE: To study the importance of glycoprotein (GP) I balpha mutation (A156V) in interaction between mutant expressing cell and immobilized vWF under fluid shear stress. METHODS: Mutant GP I balpha cDNA was cloned into the EcoR I site of the mammalian expression vector pDX, mutant cDNA was then transfected into CHO betaIX cells. Human vWF was purified from blood cryoprecipitate by glycine and NaCl precipitation and subsequent separation on sepharose 4B column. The purified vWF was immobilized onto a coverslip. Cell rolling was induced in a parallel-plate flow chamber and observed by phase-contrast video microscope. RESULTS: CHO cells expressing GP I b-IX-V complex could adhere to and roll on immobilized vWF. The A156V mutant cells retained the ability to adhere and roll on vWF matrix, but the rolling speed was significantly faster than that of wild type cells, indicating that the off-rate of the ligand-receptor bond between the mutant and vWF was impaired. Binding of monoclonal antibody AN51 to mutant GP I balpha decreased significantly, indicating that the A156V mutation altered the conformation of the N-terminal ligand-binding region of GP I balpha. CONCLUSION: The mutant GP I balpha has a faster off-rate for its interaction with immobilized vWF. The mutant polypeptide adopts an altered conformation in N-terminal ligand-binding region of GP I balpha. The parallel-plate flow chamber is an extremely useful system in evaluating interaction between GP I balpha and vWF.

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Quantitative assessment of the tension-type headache and migraine severity continuum.

Two quantitative measures for Waters' tension-type headache and migraine severity continuum are proposed. To ensure face validity, symptoms and precipitants of this disorder were complied from the literature as a basis for the Auckland Migraine and Headache Inventory. This inventory was completed by 84 participants (mean age +/- SD, 26.0 +/- 9.7 years; range, 18 to 59 years) who complied with the criteria of the International Headache Society for migraine or tension-type headache. The migraine headache index and the number of precipitants were derived from the Auckland Migraine and Headache Inventory. These scores yielded significant internal reliability (r = .77 and .84), test-retest reliability (r = .86 and .74), and concurrent validity (r = .57) coefficients. The data, therefore, support the notion that the migraine headache index and the number of precipitants are reliable and valid indices of tension-type headache and migraine severity, suitable for participant selection and assessment of treatment. This study offers support for Waters' suggestion that tension-type headache and migraine are extremes of a severity continuum.