Interaction between GPIbalpha and FcgammaIIA receptor in human platelets

Glycoprotein (GP) Ib (alpha and beta) in platelets forms a noncovalent hetero-oligomeric complex with GPIX and GPV and serves as a receptor for von Willebrand factor (vWF), which mediates the initial adhesion of platelets to the subendothelium after vascular damage and also plays a role in thrombin-induced platelet activation. We investigated the interaction between GPIbalpha and FcgammaIIA receptor using a yeast two-hybrid system and mutagenesis, and we identified residues R542G543R544 in GPIbalpha and D298D299D300 in FcgammaIIA receptor as the primary interaction sites. These results further confirmed the interaction between GPIbalpha and FcgammaIIA receptor and support the hypothesis that the signal transduction of GPIb-IX-V that leads to platelet activation may be partially mediated through FcgammaIIA receptor.     

Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX.

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.     

Regulation of von Willebrand factor binding to the platelet glycoprotein Ib-IX by a membrane skeleton-dependent inside-out signal

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb(alpha) markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.     

Agkistin, a snake venom-derived glycoprotein Ib antagonist, disrupts von Willebrand factor-endothelial cell interaction and inhibits angiogenesis

Glycoprotein (GP) Ib, an adhesion receptor expressed on both platelets and endothelial cells, mediates the binding of von Willebrand factor (vWF). Platelet GPIb plays an important role in platelet adhesion and activation, whereas the interaction of vWF and endothelial GPIb is not fully understood. We report here that agkistin, a snake venom protein, selectively blocks the interaction of vWF with human endothelial GPIb and inhibits angiogenesis in vivo. Agkistin specifically blocked human umbilical vein endothelial cell (HUVEC) adhesion to immobilized vWF in a concentration-dependent manner. Fluorescein isothiocyanate (FITC)-conjugated agkistin bound to HUVECs in a saturable manner. AP1, a monoclonal antibody (mAb) raised against GPIb, specifically inhibited the binding of FITC-conjugated agkistin to HUVECs in a dose-dependent manner, but other anti-integrin mAbs raised against alpha(v)beta(3), alpha(2)beta(1), and alpha(5)beta(1) did not affect this binding reaction. However, neither agkistin (2 microgram/ml) nor AP1 (40 microgram/ml) apparently reduced HUVEC viability. Both agkistin and AP1 exhibited a profound anti-angiogenic effect in vivo when assayed by using the 10-day-old embryo chick chorioallantoic membrane model. These results suggest endothelial GPIb plays a role in spontaneous angiogenesis in vivo, and the anti-angiogenic effect of agkistin may be because of disruption of the interaction of endogenous vWF with endothelial GPIb.     

Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets

The Mr approximately 540,000 dimeric actin gelation protein, actin-binding protein (ABP), has previously been shown in human platelets to link actin to membrane glycoprotein Ib (GPIb) (Fox, J. E. B. (1985) J. Biol. Chem. 260, 11970-11977; Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., and Kunicki, T. J. (1985) J. Cell Biol. 100, 317-321). We have examined further the interaction between ABP and GPIb. Platelet extracts were depleted of ABP by precipitation with anti-ABP monoclonal antibodies (mAbs); in resulting precipitates, ABP monomer is complexed with GPIb in a 5:1 molar ratio. The ABP.GPIb complex is resistant to chaotropic solvents but dissociated by the ionic detergent, sodium dodecyl sulfate. Treatment of intact platelets with the ionophore A23187 activates a Ca2+-dependent protease which cleaves the Mr approximately 270,000 ABP subunit into three fragments of Mr 190,000, 100,000, and 90,000; the latter fragment is derived from the Mr 100,000 fragment. Anti-ABP mAbs coprecipitated GPIb with the Mr 100,000 and 90,000 fragments, but not with the Mr 190,000 fragment which contains the ABP self-association site. In the reciprocal experiment, anti-GPIb antibodies co-precipitated only the Mr 100,000 and 90,000 ABP fragments. Actin also co-precipitated with the Mr 100,000 and 90,000, but not with the Mr 190,000 ABP fragment. The anti-ABP mAb that precipitated the Mr 100,000-90,000 GPIb-binding ABP fragment recognizes a trypsin cleavage fragment of ABP that binds actin filaments in vitro. These findings establish that both the GPIb-binding site and actin-binding sites are in the same region of the ABP monomer. Because of the extended bipolar conformation of the ABP molecule, the data suggest that the GPIb.actin-binding region is located remote from the self-association, or dimerization, site of the ABP subunit.     

Convulxin binds to native,human glycoprotein Ib alpha

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.     

Reconstitution of the platelet glycoprotein Ib-IX complex in phospholipid bilayer Nanodiscs

The glycoprotein Ib-IX (GPIb-IX) complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis via the initiation of adhesion of platelets to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge of the GPIb-IX complex was obtained from studies on platelets and transfected mammalian cells expressing the GPIb-IX complex, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that the GPIb-IX complex purified from human platelets has been reconstituted into the Nanodisc. The Nanodisc-reconstituted GPIb-IX complex was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent K(d) of 0.73 ± 0.07 nM. The binding to VWF was inhibited by anti-GPIbα antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIbβ monoclonal antibody RAM.1. Finally, the Nanodisc-reconstituted GPIb-IX complex exhibited ligand binding activity similar to that of the isolated extracellular domain of GPIbα. In conclusion, the GPIb-IX complex in Nanodiscs adopts a native-like conformation and possesses the ability to bind its natural ligands, thus making a Nanodisc a suitable in vitro platform for further investigation of this hemostatically important receptor complex.     

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.     

Characterization of a fragment of bovine von Willebrand factor that binds to platelets

Bovine von Willebrand factor was digested with human plasmin in order to isolate and characterize a fragment that can bind to human platelets. A terminal plasmin digest of bovine von Willebrand factor is composed of five fragments, ranging in relative molecular weight (Mr) from 250,000 to 35,000. The major fragment has a Mr of 250,000 and consists of four disulfide-linked polypeptide chains with Mr from 69,000 to 35,000. The Mr 69,000 and 49,000 polypeptides possess carbohydrate moieties, as indicated by their reaction with periodate-Schiff reagent. Gel filtration studies suggest that, at physiological ionic strength, four of the Mr 250,000 fragments associate into a limited noncovalent oligomer. Monoclonal antibodies were prepared against native von Willebrand factor and used to characterize the distribution of epitopes on native vWF and the Mr 250,000 major fragment. Two of the monoclonal antibodies that recognize the major fragment (2 and H-9) inhibit platelet agglutination. The Mr 250,000 fragment binds to human platelets, and the binding is inhibited by monoclonal antibodies 2 and H-9. The Mr 250,000 fragment does not agglutinate platelets, consistent with a requirement for high molecular weight oligomers of von Willebrand factor for platelet agglutination. The Mr 250,000 fragment can compete with intact, bovine von Willebrand factor for binding to human platelets. However, its affinity is one-tenth that of intact von Willebrand factor.     

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.     

Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.     

Importance of temporal flow gradients and integrin alphaIIbbeta3 mechanotransduction for shear activation of platelets

Disturbances of blood flow play an important role in promoting platelet activation and arterial thrombus formation in stenosed, injured, atherosclerotic arteries. To date, glycoprotein Ib (GPIb) has been considered the primary platelet mechanosensory receptor, responding to increased shear with enhanced adhesive and signaling function. We demonstrate here that von Willebrand factor-GPIb interaction is inefficient at inducing platelet activation even when platelets are exposed to very high wall shear stresses (60 dyn/cm(2)). Rapid platelet activation under flow was only observed under experimental conditions in which transiently adherent platelets were exposed to sudden accelerations in blood flow. Platelet responsiveness to temporal shear gradients was integrin alpha(IIb)beta(3)-dependent and occurred only on a von Willebrand factor substrate, as platelets forming integrin alpha(IIb)beta(3) adhesive contacts with immobilized fibrinogen were unresponsive to sudden increases in shear. The calcium response induced by temporal shear gradients was distinct from previously identified integrin alpha(IIb)beta(3) calcium responses in terms of its transient nature, its requirement for platelet co-stimulation by the P2Y(1) purinergic ADP receptor, and its dependence on the influx of extracellular calcium. Our studies demonstrate a key role for temporal shear gradients in promoting platelet activation. Moreover, they define for the first time the involvement of P2Y receptors in integrin mechanotransduction.     

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.     

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin alpha(IIb)beta(3)) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin alpha(IIb)beta(3). vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1, 2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.     

A CD9, alphaIIbbeta3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alphaIIbbeta3 conformational states.

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.     

Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activation both in platelets and a transfected Chinese hamster ovary cell system

In platelets, alpha(IIb)beta(3) exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert alpha(IIb)beta(3) to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute alpha(IIb)beta(3) activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which alpha(IIb)beta(3) activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected alpha(IIb)beta(3) but also endogenous alpha(v)beta(3). Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbalpha that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to alpha(IIb)beta(3) activation and suggest that the signaling pathways by which GPIb-IX induces alpha(IIb)beta(3) activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.     

The clearance mechanism of chilled blood platelets

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.     

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb(alpha)-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.     

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIb alpha in platelet-type von Willebrand disease

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib alpha (GPIb alpha), a major component of the platelet GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI alpha protein containing residues 228-238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a beta-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic 'face' consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphipathic surfaces in the 228-238 region of the Val 233-containing GPIb alpha protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.