Tetsuji Kamata

The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.

Exposure of platelets to shear stress leads to aggregation in the absence of exogenous agonists. We have now found that different adhesive proteins and platelet membrane glycoproteins are involved in aggregation depending on the shear stress conditions and the concentration of divalent cations in the medium. When blood is collected with trisodium citrate as anticoagulant, which causes a decrease in the levels of external ionized calcium ([Ca2+]o), platelet aggregation can be induced under low shear force (12 dyn/cm2) and is mediated by fibrinogen binding to the glycoprotein IIb-IIIa complex. Aggregates formed under these conditions are not stable, and when shear force is increased to 68 dyn/cm2, disaggregation results. By contrast, platelets from blood collected with hirudin as anticoagulant, wherein [Ca2+]o is within normal plasma levels, do not undergo low shear-induced aggregation; however, after exposure to a shear force above 80 dyn/cm2, aggregation is observed but only when von Willebrand factor is present and can interact with both its platelet binding sites, glycoprotein Ib-IX and glycoprotein IIb-IIIa. Fibrinogen is not involved in high shear-induced aggregation which, in fact, occurs normally in patients with severe afibrinogenemia. Thus, von Willebrand factor in the absence of exogenous agonists can mediate platelet aggregation in experimental conditions that may mimic the hemorheological situation of partially occluded arteries. This pathway of platelet aggregation involving only one adhesive ligand and two membrane adhesion receptors may play a relevant role in thrombogenesis.

Identification of putative ligand binding sites within I domain of integrin α2β1(VLA-2, CD49b/CD29)

Integrin alpha 2 beta 1 is a cell surface adhesion receptor for collagen and echovirus 1. Here we localized the epitopes for anti-alpha 2 monoclonal antibodies using interspecies (human/bovine) alpha 2 chimeras with different lengths of human alpha 2 sequence on the amino-terminal side and site-directed mutagenesis. The antibodies that block the collagen and/or echovirus 1 binding to human alpha 2 beta 1 (6F1, RMAC11, 12F1, and AA10) recognizes a small region (residues 173-259) within the I domain. Asp-160 and Arg-242 are critical for binding of the two other function-inhibiting antibodies, P1H5 and 5E8, respectively. Notably, mutations of Asp-151 and Asp-254 block the binding of alpha 2 beta 1 to collagen. These data suggest that the I domain (residues 140-359) is critically involved in the ligand/receptor interactions, and collagen and echovirus 1 binding sites are adjacent or overlapping within the I domain. The sequence of the residues 173-259 of alpha 2 overlap with the peptide sequences (M11 and M20) that derive from von Willebrand factor A1 and A3 domains (homologous to the alpha 2 I domain) and block von Willebrand factor/collagen interaction, suggesting that the epitope region of alpha 2 (residues 173-259) may really be involved in ligand recognition.

Critical amino acid residues for ligand binding are clustered in a predicted β-turn of the third N-terminal repeat in the integrin α4 and α5 subunits

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)‐1 and fibronectin (CS‐1). The alpha 4 beta 1‐ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine‐scanning mutagenesis of the previously localized putative ligand binding sites (residues 108–268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM‐1 and CS‐1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta‐turn structure (residues 181–190) of the third N‐terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin‐alpha 5 beta 1 interaction. These findings suggest that the predicted beta‐turn structure could be ubiquitously involved in ligand binding of non‐I domain integrins.

Critical Residues of Integrin αIIb Subunit for Binding of αIIbβ3 (Glycoprotein IIb-IIIa) to Fibrinogen and Ligand-mimetic Antibodies (PAC-1, OP-G2, and LJ-CP3)

Integrin αIIbβ3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of αIIbβ3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of αIIb significantly block binding of αIIbβ3 to soluble fibrinogen. These mutations also block binding of αIIbβ3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of αIIbβ3, in contrast to most of the natural αIIb mutations occurring in Glanzmanns thrombasthenic patients. The data suggest that these residues are critically involved in αIIbβ3-ligand interactions.

The Role of the CPNKEKEC Sequence in the β2 Subunit I Domain in Regulation of Integrin αLβ2 (LFA-1)

The αL I (inserted or interactive) domain of integrin αLβ2 undergoes conformational changes upon activation. Recent studies show that the isolated, activated αL I domain is sufficient for strong ligand binding, suggesting the β2 subunit to be only indirectly involved. It has been unclear whether the activity of the αL I domain is regulated by the β2 subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169–176) in the β2 I domain with a corresponding β3 sequence, or mutating Lys174 to Thr, constitutively activates αLβ2 binding to ICAM-1. These mutants do not require Mn2+ for ICAM-1 binding and are insensitive to the inhibitory effect of Ca2+. We have also localized a component of the mAb 24 epitope (a reporter of β2 integrin activation) in the CPNKEKEC sequence. Glu173 and Glu175 of the β2 I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon β2 integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the αL I domain did not eliminate the mAb 24 epitope. This confirms that the αL I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the β2 subunit I domain. These results suggest that the CPNKEKEC sequence of the β2 I domain is involved in regulating the αL I domain.

Critical cysteine residues for regulation of integrin αIIbβ3 are clustered in the epidermal growth factor domains of the β3 subunit

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin beta3 subunit are involved in the regulation of alphaIIbbeta3. In the present study, we studied systematically the role of disulphide bonds in beta3 on the ligand-binding function of alphaIIbbeta3 by mutating individual cysteine residues of beta3 to serine. We found that the disulphide bonds that are critical for alphaIIbbeta3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate alphaIIbbeta3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated alphaIIbbeta3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep alphaIIbbeta3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.

Interaction between collagen and the α2 I-domain of integrin αβ1: Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region

A docking model of the α2 I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512–28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of α2 (residues 284–288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole α2β1, with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. α1, α2, and α10) bind to collagen in a similar fashion.

Echovirus 1 interaction with the human very late antigen-2 (integrin α2β1) I domain: Identification of two independent virus contact sites distinct from the metal ion-dependent adhesion site

The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the α2 subunit of the VLA-2 α2β1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199–201 and 212–216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.

Structural Requirements for Activation in αIIbβ3 Integrin

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.

Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele‐specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)‐289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)‐324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)‐747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αIIbβ3 surface expression, the wild‐type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild‐type β3 cDNA. Flow cytometric analysis using an anti‐αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild‐type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3, αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.