Kinetics of GPIbalpha-vWF-A1 tether bond under flow: effect of GPIbalpha mutations on the association and dissociation rates

The interaction between platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor (vWF) is the first step of the hemostatic response to vessel injury. In platelet-type von Willebrand disease, two mutations, G233V and M239V, have been described within the Cys209-Cys248 disulfide loop of GPIbalpha that compromise hemostasis by increasing the affinity for vWF. We have earlier shown that converting other residues in this region to valine alters the affinity of GPIbalpha for vWF, with mutations K237V and Q232V, respectively, showing the greatest increase and decrease in affinity. Here, we investigated further the effect of these two mutations on the kinetics of the GPIbalpha interaction with the vWF-A1 domain under dynamic flow conditions. We measured the cellular on- and off-rate constants of Chinese hamster ovary cells expressing GPIb-IX complexes containing wild-type or mutant GPIbalpha interacting with vWF-A1-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on vWF-A1 surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of vWF-A1, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency, was threefold more and threefold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GPIbalpha affect both the association and dissociation kinetics of the GPIbalpha-vWF-A1 bond. These findings are in contrast to the functionally similar selectin bonds where some of the mutations have been reported to affect only the dissociation rate.     

The snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation

Botrocetin is a snake venom protein that enhances the affinity of the A1 domain of plasma von Willebrand factor (vWF) for the platelet receptor glycoprotein Ibalpha (GPIbalpha), an event that contributes to bleeding and host death. Here we describe a kinetic and crystallographic analysis of this interaction that reveals a novel mechanism of affinity enhancement. Using high-temporal-resolution microscopy, we show that botrocetin decreases the GPIbalpha off-rate two-fold in both human and mouse complexes without affecting the on-rate. The key to this behavior is that, upon binding of GPIbalpha to vWF-A1, botrocetin prebound to vWF-A1 makes no contacts initially with GPIbalpha, but subsequently slides around the A1 surface to form a new interface. This two-step mechanism and flexible coupling may prevent adverse alterations in on-rate of GPIbalpha for vWF-A1, and permit adaptation to structural differences in GPIbalpha and vWF in several prey species.     

Shielding of the A1 domain by the D'D3 domains of von Willebrand factor modulates its interaction with platelet glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.     

The cytoplasmic domain of the platelet glycoprotein Ibalpha is phosphorylated at serine 609

The alpha chain of the platelet von Willebrand factor receptor, glycoprotein (GP) Ib, is not known to be phosphorylated. Here, we report that the cytoplasmic domain of GPIbalpha is phosphorylated at Ser(609); this was detected by immunoblotting with an anti-phosphopeptide antibody, anti-pS609, that specifically recognizes the GPIbalpha C-terminal sequence S(606)GHSL(610) only when Ser(609) is phosphorylated. Immunoabsorption with anti-pS609 removed almost all of the GPIbalpha from platelet lysates, indicating a high proportion of GPIbalpha phosphorylation. Anti-pS609 inhibited GPIb-IX binding to the intracellular signaling molecule, 14-3-3zeta. Dephosphorylation of GPIb-IX with potato acid phosphatase inhibited anti-pS609 binding and also 14-3-3zeta binding. A synthetic phosphopeptide corresponding to the GPIbalpha C-terminal sequence (SIRYSGHpSL), but not a nonphosphorylated identical peptide, abolished GPIb-IX binding to 14-3-3zeta. Thus, phosphorylation at Ser(609) of GPIbalpha is important for 14-3-3zeta binding to GPIb-IX. In certain regions of spreading platelets, particularly at the periphery, there was a reduction in GPIbalpha staining by anti-pS609 as observed under a confocal microscope, indicating that a subpopulation of GPIbalpha molecules in these regions is dephosphorylated. These data suggest that phosphorylation and dephosphorylation at Ser(609) of GPIbalpha regulates GPIb-IX interaction with 14-3-3 and may play important roles in the process of platelet adhesion and spreading.     

Interaction between platelet glycoprotein Ibalpha and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear.

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIbalpha and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIbalpha intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569-579 of the GPIbalpha cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant Delta557-568 and Delta569-579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIbalpha for interaction with filamin-1.     

The linker between the D3 and A1 domains of vWF suppresses A1‐GPIbα catch bonds by site‐specific binding to the A1 domain

Platelet attachment to von Willebrand factor (vWF) requires the interaction between the platelet GP1bα and exposed vWF-A1 domains. Structural insights into the mechanism of the A1-GP1bα interaction have been limited to an N-terminally truncated A1 domain that lacks residues Q₁₂₃₈ − E₁₂₆₀ that make up the linker between the D3 and A1 domains of vWF. We have demonstrated that removal of these residues destabilizes quaternary interactions in the A1A2A3 tridomain and contributes to platelet activation under high shear (Auton et al., J Biol Chem 2012;287:14579–14585). In this study, we demonstrate that removal of these residues from the single A1 domain enhances platelet pause times on immobilized A1 under rheological shear. A rigorous comparison between the truncated A1-1261 and full length A1-1238 domains demonstrates a kinetic stabilization of the A1 domain induced by these N-terminal residues as evident in the enthalpy of the unfolding transition. This stabilization occurs through site and sequence-specific binding of the N-terminal peptide to A1. Binding of free N-terminal peptide to A1-1261 has an affinity and this binding although free to dissociate is sufficient to suppress the platelet pause times to levels comparable to A1-1238 under shear stress. Our results support a dual-structure/function role for this linker region involving a conformational equilibria that maintains quaternary A domain associations in the inactive state of vWF at low shear and an intra-A1-domain conformation that regulates the strength of platelet GP1bα-vWF A1 domain associations in the active state of vWF at high shear.     

High molecular weight kininogen regulates platelet-leukocyte interactions by bridging Mac-1 and glycoprotein Ib

Leukocyte-platelet interaction is important in mediating leukocyte adhesion to a thrombus and leukocyte recruitment to a site of vascular injury. This interaction is mediated at least in part by the beta2-integrin Mac-1 (CD11b/CD18) and its counter-receptor on platelets, glycoprotein Ibalpha (GPIbalpha). High molecular weight kininogen (HK) was previously shown to interact with both GPIbalpha and Mac-1 through its domains 3 and 5, respectively. In this study we investigated the ability of HK to interfere with the leukocyte-platelet interaction. In a purified system, HK binding to GPIbalpha was inhibited by HK domain 3 and the monoclonal antibody (mAb) SZ2, directed against the epitope 269-282 of GPIbalpha, whereas mAb AP1, directed to the region 201-268 of GPIbalpha had no effect. In contrast, mAb AP1 inhibited the Mac-1-GPIbalpha interaction. Binding of GPIbalpha to Mac-1 was enhanced 2-fold by HK. This effect of HK was abrogated in the presence of HK domains 3 or 5 or peptides from the 475-497 region of the carboxyl terminus of domain 5 as well as in the presence of mAb SZ2 but not mAb AP1. Whereas no difference in the affinity of the Mac-1-GPIbalpha interaction was observed in the absence or presence of HK, maximal binding of GPIbalpha to Mac-1 doubled in the presence of HK. Moreover, HK/HKa increased the Mac-1-dependent adhesion of myelomonocytic U937 cells and K562 cells transfected with Mac-1 to immobilized GPIbalpha or to GPIbalpha-transfected Chinese hamster ovary cells. Finally, Mac-1-dependent adhesion of neutrophils to surface-adherent platelets was enhanced by HK. Thus, HK can bridge leukocytes with platelets by interacting via its domain 3 with GPIbalpha and via its domain 5 with Mac-1 thereby augmenting the Mac-1-GPIbalpha interaction. These distinct molecular interactions of HK with leukocytes and platelets contribute to the regulation of the adhesive behavior of vascular cells and provide novel molecular targets for reducing atherothrombotic pathologies.     

Functional analysis of a recombinant glycoprotein Ia/IIa (Integrin alpha(2)beta(1)) I domain that inhibits platelet adhesion to collagen and endothelial matrix under flow conditions

The interaction of platelets with collagen plays an important role in primary hemostasis. Glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) is a major platelet receptor for collagen. The binding site for collagen has been mapped to the I domain within the alpha(2) subunit (GPIa). In order to assess the role of the alpha(2)-I domain structure in GPIa/IIa binding to collagen, a recombinant I domain (amino acids 126-337) was expressed in Escherichia coli. The alpha(2)-I protein bound human types I and III collagen in a saturable and divalent cation-dependent manner and was blocked by the alpha(2)beta(1) function blocking antibody 6F1. The alpha(2)-I protein inhibited collagen-induced platelet aggregation (IC(50) = 600 nM). Unexpectedly, 6F1, an antibody that fails to inhibit platelet aggregation in platelet-rich plasma, blocked the inhibitory effect of the alpha(2)-I protein. The alpha(2)-I protein was able to prevent platelet adhesion to a collagen surface exposed to flowing blood under low shear stress. Interestingly, it inhibited platelet adhesion to extracellular matrix at high shear stress. These results, taken together, provide firm evidence that GPIa/IIa directly mediates the first contact of platelets with collagen under both stirring and flow conditions.     

人von Willebrand因子A1和A3功能结构域嵌合体的表达及生物学功能

vWF介导血小板粘附到细胞外基质 ,在血栓形成过程中发挥重要作用 ,可通过阻断vWF与细胞外基质的结合阻止血小板的粘附 .应用RT PCR方法从人脐静脉内皮细胞中克隆vWF分子A1、A3区基因并在大肠杆菌中表达 ,表达的重组蛋白量占菌体总蛋白 12 6 % ,包涵体经过变性剂溶解、纯化和复性 ,获得重组蛋白 (rvWF A1 A3) .应用流式细胞术检测rvWF A1 A3与血小板膜糖蛋白 (GPⅠb)的结合功能 ;血小板聚集实验观察rvWF A1 A3对瑞斯托霉素 (ristocetin)诱导的血小板聚集 (RIPA)的影响 ;ELISA胶原结合实验及竞争抑制实验分析rvWF A1 A3与胶原的结合活性 .结果显示 :rvWF A1 A3嵌合体与血小板的结合阳性率为 70 4 % .rvWF A1 A3嵌合体不能引起血小板的聚集 ,但rvWF A1 A3嵌合体与血小板温育后可以阻断ristocetin诱导人血浆vWF对血小板的聚集作用 ,而且呈剂量依赖性 ,IC50 的rvWF A1 A3浓度为 0 76 μmol L ,当浓度为 1 17μmol L时抑制率最高达 76 8% .rvWF A1 A3具有良好的胶原结合活性 ,同时它可以竞争性抑制vWF与Ⅲ型胶原的结合 ,抑制率为 76 % .表明rvWF A1 A3可作为阻断剂用于干预vWF介导的血小板粘附过程 ,同时又可以阻断血浆vWF与血小板GPIb结合抑制血小板聚集 ,具有良好的抗栓应用前景 .     

Identification of a binding site for glycoprotein Ibalpha in the Apple 3 domain of factor XI

Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg(369)-Ile(370) bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (K(d) = approximately 10 nM, B(max) = approximately 1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn(2+)) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI. Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbalpha, in a Zn(2+)-dependent fashion (K(d) = approximately 52 nM). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn(2+), whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn(2+), glycocalicin binds FXI at a heparin-binding site in A3 (Lys(252) and Lys(253)) and not by amino acids previously shown to be required for platelet binding (Ser(248), Arg(250), Lys(255), Phe(260), and Gln(263)). However, binding in the presence of HK and Zn(2+) requires Ser(248), Arg(250), Lys(255), Phe(260), and GLn(263) and not Lys(252) and Lys(253). Thus, binding of FXI to GPIbalpha is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.     

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.     

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.     

Thrombospondin-bound integrin-associated protein (CD47) physically and functionally modifies integrin alphaIIbbeta3 by its extracellular domain

Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, alphaIIbbeta3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to alphaIIbbeta3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the alphaIIbbeta3 with mutated beta3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type alphaIIbbeta3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of alphaIIbbeta3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to alphaIIbbeta3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified alphaIIbbeta3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with alphaIIbbeta3 and can change alphaIIbbeta3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin.     

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15‐mer random library on the outer surface of Escherichia coli (E.coli), high‐affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On‐cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off‐cell, using peptide‐ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB‐binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The K_d measured by peptide‐ELISA off‐cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.     

Role of the C2 domain of factor VIIIa in the assembly of factor-X activating complex on the platelet membrane

Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.     

Design of peptides with high affinities for heparin and endothelial cell proteoglycans

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.     

Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha regulate shear-dependent cell adhesion to von Willebrand factor

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbalpha (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbalpha, cells expressing GPIbalpha, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s(-1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.     

GPIbα-vWF rolling under shear stress shows differences between type 2B and 2M von Willebrand disease

Both type 2B and type 2M von Willebrand disease result in bleeding disorders; however, whereas type 2B has increased binding affinity between platelet glycoprotein Ibα and von Willebrand factor (vWF), type 2M has decreased binding affinity between these two molecules. We used R687E type 2B and G561S type 2M vWF-A1 mutations to study binding between flowing platelets and insolubilized vWF mutants. We measured rolling velocities, mean stop times, and mean go times at 37°C using high-speed video microscopy. The rolling velocities for wt-wt interactions first decrease, reach a minimum, and then increase with increasing shear stress, indicating a catch-slip transition. By changing the viscosity, we were able to quantify the effects of force versus shear rate for rolling velocities and mean stop times. Platelet interactions with loss-of-function vWF-A1 retain the catch-slip bond transition seen in wt-wt interactions, but at a higher shear stress compared with the wt-wt transition. The mean stop time for all vWF-A1 molecules reveals catch-slip transitions at different shear stresses (gain-of-function vWF-A1 < wt vWF-A1< loss-of-function vWF-A1). The shift in the catch-slip transition may indicate changes in how the different mutants become conformationally active, indicating different mechanisms leading to similar bleeding characteristics.     

Identification of the C3b binding site in a recombinant vWF-A domain of complement factor B by surface-enhanced laser desorption-ionisation affinity mass spectrometry and homology modelling: implications for the activity of factor B

Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH3) in the presence of Mg(2+). A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central beta-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A beta-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.     

4-Fluoroproline derivative peptides: effect on PPII conformation and SH3 affinity.

Eukaryotic signal transduction involves the assembly of transient protein-protein complexes mediated by modular interaction domains. Specific Pro-rich sequences with the consensus core motif PxxP adopt the PPII helix conformation upon binding to SH3 domains. For short Pro-rich peptides, little or no ordered secondary structure is usually observed before binding interactions. The association of a Pro-rich peptide with the SH3 domain involves unfavorable binding entropy due to the loss of rotational freedom on forming the PPII helix. With the aim of stabilizing the PPII helix conformation in the Pro-rich HPK1 decapeptide PPPLPPKPKF (P2), a series of P2 analogues was prepared, in which specific Pro positions were alternatively occupied by 4(S)- or 4(R)-4-fluoro-L-proline. The interactions of these peptides with the SH3 domain of the HPK1-binding partner HS1 were quantitatively analyzed by the NILIA-CD approach. A CD thermal analysis of the P2 analogues was performed to assess their propensity to adopt the PPII helix conformation. Contrary to our expectations, the K(d) values of the analogues were lower than that of the parent peptide P2. These results clearly show that the induction of a stable PPII helix conformation in short Pro-rich peptides is not sufficient to increase their affinity toward the SH3 domain and that the effect of 4-fluoroproline strongly depends on the position of this residue in the sequence and the chirality of the substituent in the pyrrolidine ring.