Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.     

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.     

Ability of Different Glycoprotein IIb-IIIa Ligands to Support Platelet Aggregation Induced by Activating Antibody CRC54

The ability of different ligands of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3-integrin) to support platelet aggregation stimulated by activating anti-GP IIb-IIIa monoclonal antibody (monoAB) CRC54 has been investigated. Antibody CRC54 stimulated aggregation of washed platelets not only in the presence of fibrinogen, the main GP IIb-IIIa ligand, but also in the presence of von Willebrand factor (vWF). Unlike these ligands, fibronectin failed to support CRC54-induced aggregation. Fibrinogen and vWF dependent platelet aggregation was completely suppressed by GP IIb-IIIa antagonists--preparations Monafram (F(ab')2 fragments of monoAB that blocked GP IIb-IIIa receptor activity) and aggrastat (RGD-like peptidomimetic). However, aggregation stimulated in the presence of vWF was also completely inhibited by monoAB AK2 directed against GP Ib and capable of blocking its binding with vWF. CRC54-induced aggregation of platelets from patient with GP Ib deficiency in the presence of vWF was significantly lower than aggregation of platelets from normal donors and was not inhibited by anti-GP Ib antibody but still blocked by GP IIb-IIIa antagonist Monafram. Monafram also suppressed CRC54-stimulated platelet adhesion to plastic-adsorbed fibrinogen, vWF, and fibronectin. Unlike CRC54-induced platelet aggregation supported by fluid phase vWF, CRC54-induced adhesion to adsorbed vWF was not affected by anti-GP Ib antibody. Aggregation induced by CRC54 in the presence of fibrinogen and vWF was only partially suppressed by prostaglandin E1, an inhibitor of platelet activation, and was associated with serotonin release from platelet granules only when Ca2+ concentration was decreased from 1 mM (physiological level) to 0.1 mM. The data indicate that vWF supports CRC54-induced platelet aggregation via interaction with two receptors--GP IIb-IIIa and GP Ib. Aggregation induced by CRC54 in the presence of vWF or fibrinogen is only partially dependent on platelet activation and is accompanied with granule secretion only at low Ca2+ concentrations.     

Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane

Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (means +/- SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib alpha (22 000 +/- 2700, n = 3), WM 23, epitope on GP Ib alpha (21 000 +/- 3400, n = 3), FMC 25, epitope on GP IX (20 100 +/- 2700, n = 3), and FMC 25 (Fab')2 (27 100 +/- 800, n = 2).(ABSTRACT TRUNCATED AT 400 WORDS)     

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.     

A critical evaluation of the available methods for the determination of factor VIII von Willebrand

Von Willebrand factor (vWf) is the major component of the circulating factor VIII complex. The von Willebrand molecule includes factor VIII related antigen (VIIIR: Ag) which represents the molecular substrate of the von Willebrand activity expressed as Ristocetin cofactor (VIIIR:RCoF) activity. Several methods have been developed for VIIIR: Ag evaluation, among the first being the rocket-immunoelectrophoresis method of LAURELL. Radial immunodiffusion (MANCINI's method) was also used. Subsequently, radioimmunological assays, either as radioimmunoassay (RIA) or immunoradiometric assay (IRMA), were developed with improvements in sensitivity, so that levels of VIIIR: Ag lower than 0.1% of normal can be detected. More recently, an enzyme-linked immunosorbent assay (ELISA), characterized by the use of enzyme-conjugated antibody was proposed. This method shows a sensitivity similar to immunoradiometric methods but without using any dangerous reagent. Finally, a nephelometric method was proposed for factor VIII antigen evaluation. For a qualitative evaluation of von Willebrand factor crossed-immunoelectrophoresis and multimeric analysis can be used. In the first case, the use of precipiting antibodies against von Willebrand factor may demonstrate a peak with different characteristics related to the biochemical property of von Willebrand. Multimeric analysis in SDS-agarose gel electrophoresis followed by staining with labelled antifactor VIII antibodies gives information about different polymeric forms of circulating VIII/vW factor. Von Willebrand factor activity, expressed as its ability to induce platelet aggregation in the presence of the antibiotic Ristocetin, can be carried out using normal formalin fixed platelets, either with aggregometer or visual methods (glass slide test or tubes test and microtritation plate). The corrected evaluation of factor VIII complex by all these techniques together with the clotting activity assay allows a satisfactory study of factor VIII properties.     

Modulation of platelet function through adhesion receptors. A dual role for glycoprotein IIb-IIIa (integrin alpha IIb beta 3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor.

We have found that the form of glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) expressed on nonstimulated platelets is a functional receptor that mediates selective and irreversible adhesion to immobilized fibrinogen. This occurs even in the presence of the elevated intracellular cAMP levels induced by prostaglandin E1 or after inhibition of protein kinase C activity by sphingosine. In the absence of inhibitors, platelets adhering to fibrinogen through GP IIb-IIIa become fully activated and aggregate with one another. Immobilized von Willebrand factor (vWF), in contrast, is recognized by nonstimulated platelets through another receptor, GP Ib. This interaction leads to a change in the ligand recognition specificity of GP IIb-IIIa that can then bind to immobilized vWF and mediate irreversible platelet adhesion and aggregation; this process, however, is inhibited by elevated intracellular cAMP levels or blockade of protein kinase C activity. Therefore, GP Ib and GP IIb-IIIa induce platelet activation through the selective recognition of immobilized vWF and fibrinogen, respectively, in the absence of exogenous agonists. Moreover, "nonactivated" and "activated" GP IIb-IIIa exhibits distinctly different reactivity toward surface-bound vWF, and the functional switch can be induced by the binding of vWF to GP Ib. These findings demonstrate the modulation of platelet function by two different adhesion receptors, GP Ib and GP IIb-IIIa, as well as the distinct dual role of the latter as the necessary common mediator of irreversible adhesion and aggregation on both fibrinogen and vWF.     

Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex

Whether the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor for ristocetin-dependent binding of von Willebrand factor (vWF) has been examined by reconstitution with the purified components using a solid-phase bead assay. Purified GP Ib-IX complex was bound and orientated on the beads via a monoclonal antibody, FMC 25, directed against the membrane-associated region of the complex. Specific binding of 125I-labeled vWF to the GP Ib-IX complex coated beads was strictly ristocetin dependent with maximal binding occurring at ristocetin concentrations greater than or equal to 1 mg/mL. Ristocetin-dependent specific binding of 125I-labeled vWF was saturable. The observed binding was specific to the interaction between vWF and the GP Ib-IX complex since there was no ristocetin-dependent specific binding of vWF if the physicochemically related platelet membrane glycoprotein, GP IIb, was substituted for the GP Ib-IX complex in a corresponding bead assay. Further, neither bovine serum albumin nor other adhesive glycoproteins, such as fibrinogen or fibronectin, specifically bound to the GP Ib-IX complex in the presence of ristocetin. Ristocetin-dependent binding of vWF to platelets and to GP Ib-IX complex coated beads was inhibited by monoclonal antibodies against a 45,000 molecular weight N-terminal region of GP Ib but not by monoclonal antibodies directed against other regions of the GP Ib-IX complex. Similar correspondence between platelets and purified GP Ib-IX complex with respect to the ristocetin-dependent binding of vWF was obtained with anti-vWF monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mean platelet volume: Interrelation with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (αIIb/β3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) have been investigated in this study. The study was performed in a group of healthy volunteers (n = 38) and a group of patients with acute coronary syndrome (ACS, n = 116). Patient’s blood was collected at days 1, 3–5 and 8–12 after ACS development. All patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) as the antiaggregant therapy and most of them also received clopidogrel (ADP receptor antagonist), except 44 patients who had not taken clopidogrel at day 1 before first blood collection. Aggregation of volunteers’ platelets was stimulated by 1.25, 2.5, 5 and 20 μM ADP, while aggregation of patients’ platelets was stimulated by 5 and 20 μM ADP. GP IIb-IIIa and GP Ib content on the platelet surface was measured using ¹²⁵I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels have been recognized at 1.25 μM and 2.5 μM ADP (correlation coefficient (r) values of 0.396 and 0.373, p < 0.05), while at 5 μM and 20 μM ADP these interactions did not reach the level of statistical significance (r values of 0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients receiving ASA but before the beginning of clopidogrel treatment (r values of 0.526, p < 0.001 and 0.368, p < 0.05 for 5 and 20 μM ADP, respectively). Correlations between these parameters were not found during combined treatment of patients with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in both healthy donors and ACS patients (at all time points): the r values ranged from 0.439 to 0.647 (p ≤ 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((?5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicate that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.     

Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages

The glycoprotein (GP) Ib-IX complex is a major component of the platelet membrane which mediates adhesion of platelets to exposed subendothelium. GP Ib is a heterodimer with a large alpha chain (Mr = 135,000-145,000) and small beta chain (Mr = 22,000-27,000) linked by a disulfide bond(s). GP Ib is bound in a noncovalent 1:1 complex with GP IX (Mr = 17,000-22,000). We labeled isolated human platelets with [3H] palmitate or surface-labeled platelet membrane glycoproteins with sodium periodate-[3H]sodium borohydride and immunoprecipitated the GP Ib-IX complex from radiolabeled platelet lysates using a mouse monoclonal antibody (SZ.1) which recognizes the intact complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates from [3H]palmitate-labeled platelets revealed two radiolabeled bands under reducing conditions at 24 and 19 kDa and two bands under nonreducing conditions at 170 and 19 kDa. As demonstrated by the parallel analysis of immunoprecipitates from periodate-[3H]sodium borohydride-labeled platelets, the [3H]palmitate-labeled bands obtained under reducing conditions corresponded to GP Ib beta and GP IX and the ones obtained under nonreducing conditions to intact GP Ib and GP IX, respectively. Using alkaline methanolysis followed by high pressure liquid chromatography analysis of the methanolysis products, we demonstrated that the radioactivity associated with the GP Ib-IX complex from [3H]palmitate-labeled platelets was, in fact, covalently bound [3H]palmitate in ester linkage to protein. The protein-fatty acid linkage was also disrupted by hydroxylamine at neutral pH. Thus, this study demonstrates that GP Ib beta and GP IX in human platelets are both fatty acid-acylated with palmitate through thioester linkages.     

Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity

Activation of human platelets by complement proteins C5b-9 is accompanied by the release of small plasma membrane vesicles (microparticles) that are highly enriched in binding sites for coagulation factor Va and exhibit prothrombinase activity. We have now examined whether assembly of the prothrombinase enzyme complex (factors VaXa) is directly linked to the process of microparticle formation. Gel-filtered platelets were incubated without stirring with various agonists at 37 degrees C, and the functional expression of cell surface receptors on platelets and on shed microparticles was analyzed using specific monoclonal antibodies and fluorescence-gated flow cytometry. In addition to the C5b-9 proteins, thrombin, collagen, and the calcium ionophore A23187 were each found to induce formation of platelet microparticles that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa. These microparticles were enriched in binding sites for factor Va, and their formation paralleled the expression of catalytic surface for the prothrombinase enzyme complex. Little or no microparticle release or prothrombinase activity were observed when platelets were stimulated with epinephrine and ADP, despite exposure of platelet fibrinogen receptors by these agonists. When platelets were exposed to thrombin plus collagen, the shed microparticles contained activated GP IIb-IIIa complexes that bound fibrinogen. By contrast, GP IIb-IIIa incorporated into C5b-9 induced microparticles did not express fibrinogen receptor function. Platelets from a patient with an isolated defect in inducible procoagulant activity (Scott syndrome) were found to be markedly impaired in their capacity to generate microparticles in response to all platelet activators, and this was accompanied by a comparable decrease in the number and function of inducible factor Va receptors. Taken together, these data indicate that the exposure of the platelet factor Va receptor is directly coupled to plasma membrane vesiculation and that this event can be dissociated from other activation-dependent platelet responses. Since a catalytic membrane surface is required for optimal thrombin generation, platelet microparticle formation may play a role in the normal hemostatic response to vascular injury.     

Cyclic AMP-dependent phosphorylation of glycoprotein Ib inhibits collagen-induced polymerization of actin in platelets

Platelet function is inhibited by agents such as prostaglandin E1 (PGE1) that elevate the cytoplasmic concentration of cyclic AMP. Inhibition presumably results from the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are actin-binding protein, P51 (Mr = 51,000), P36 (Mr = 36,000), P24 (Mr = 24,000), and P22 (Mr = 22,000). Recently, we identified P24 as the beta-chain of glycoprotein (GP) Ib, a component of the plasma membrane GP Ib.IX complex. The existence of Bernard-Soulier syndrome, a hereditary disorder in which platelets selectively lack the GP Ib.IX complex, enabled us to examine whether the phosphorylation of GP Ib beta (P24) is responsible for any of the inhibitory effects of elevated cyclic AMP on platelet function. Exposure of control platelets to PGE1 increased phosphorylation of actin-binding protein, P51, P36, GP Ib beta, and P22. Prostaglandin E1 induced the same phosphorylation reactions in Bernard-Soulier platelets, except that of GP Ib beta, which is absent. In control platelets, PGE1 inhibited collagen-induced phosphorylation of myosin light chain, phosphorylation of P47 (an unidentified Mr 47,000 cytoplasmic protein that is phosphorylated by protein kinase C in stimulated platelets), aggregation, and the secretion of granule contents. Despite the absence of GP Ib beta, PGE1 also inhibited these collagen-induced responses in Bernard-Soulier platelets. However, while PGE1 inhibited collagen-induced polymerization of actin in control platelets, it did not inhibit actin polymerization in Bernard-Soulier platelets. These results suggest that cyclic AMP-induced phosphorylation of GP Ib inhibits collagen-induced actin polymerization in platelets. Because actin polymerization is required for at least some of the functional responses of platelets to an agonist, phosphorylation of Gp Ib beta may be one way in which cyclic AMP inhibits platelet function.     

Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex

Platelet activation converts the membrane GP IIb-IIIa complex into a functional receptor for fibrinogen, but the mechanism is poorly understood. We asked whether induction of receptor competency coincides with a conformational change affecting the spatial arrangement of exoplasmic domains of the IIb and IIIa subunits. Epitopes on these subunits were labeled with monoclonal antibodies conjugated to either a donor fluorescein (FITC) or an acceptor tetramethylrhodamine (TR) chromophore. Then, fluorescence resonance energy transfer (RET) between platelet-bound FITC and TR was measured by flow cytometry. In unstimulated platelets, 6-8% RET efficiency was detected between antibody B1B5, bound to GP IIb, and antibody SSA6, bound to GP IIIa, regardless of which antibody served as RET donor. RET was also observed between these antibodies and A2A9, an antibody specific for the GP IIb-IIIa complex. Cell stimulation by thrombin, ADP plus epinephrine or phorbol-ester caused up to a 2-fold increase in RET between chromophore-labeled, platelet-bound B1B5, SSA6, and A2A9 (p less than or equal to 0.05), suggesting a change in the separation or orientation of these epitopes within the GP IIb-IIIa complex. The activation-related conformational change detected by the increase in RET between antibody B1B5 and SSA6 was independent of receptor occupancy since it was unaffected by the addition of fibrinogen or by the inhibition of fibrinogen binding by the antibody, A2A9, or the peptide, RGDS. In contrast to these results with antibodies bound to different epitopes within GP IIb-IIIa, no RET was observed between FITC-A2A9 and TR-A2A9 bound to different GP IIb-IIIa complexes or between a TR-labeled GP Ib antibody and FITC-labeled GP IIb-IIIa antibodies. These studies demonstrate that platelet activation causes a change in the spatial separation or orientation of exoplasmic domains within GP IIb and IIIa, which may serve to convert this integrin into a functional adhesion receptor.     

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.     

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper), stimulates platelets by binding to α2β1 integrin and glycoprotein Ib, activating Syk and phospholipase Cγ 2, but does not involve the glycoprotein VI/Fc receptor γ chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.     

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa

Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprotein Ib and the IIb/IIIa complex. Combination of antibody to Ib and antibody to the IIb/IIIa complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins Ib and the IIb/IIIa complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.     

The immunodiagnosis of thrombocyte membrane glycoprotein deficiencies: Glanzmann's thrombasthenia, Bernard-Soulier syndrome and GMP-140 protein deficiency]

Immunological methods were developed for the diagnosis of platelet membrane glycoprotein (GP) deficiencies. The number of membrane GP on platelet surface was determined as the binding of 125I-labeled monoclonal antibodies (mAB) directed against individual platelet GP. Total amount of GP in platelet lysate was assessed by immunoblotting with specific polyclonal antibodies. Methods were applied for patients with different thrombocytopathies. Binding of mAB VM16a, directed against GP IIb-IIIa was strongly decreased in patients with Glanzmann's thrombasthenia (0.5-14.5% of normal) and binding of anti-GP Ib mAB VM16d--in patient with Bernard-Soulier syndrome (0.5% of control) indicating the deficiencies of corresponding GP. In patient with gray platelet syndrome binding of both antibodies was not decreased but even increased. It was shown by immunoblotting that platelets from the patient with gray platelet syndrome contained normal amount of GP IIa, but strongly decreased amount of GMP-140 (14.5% of control)--membrane GP of platelet--granules.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase

Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib.     

Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding.

We have expressed in mammalian cells a fragment (residues 1-302) of the alpha chain of platelet glycoprotein (GP) Ib containing the von Willebrand factor- (vWF) binding site. The secreted soluble protein had an apparent molecular mass of 45 kDa and reacted with conformation-dependent monoclonal antibodies that bind only to native GP Ib, thus demonstrating its proper folding. After insolubilization on nitrocellulose membrane, the recombinant GP Ib alpha fragment bound soluble vWF in the presence of ristocetin or botrocetin with a dissociation constant similar to that exhibited by GP Ib.IX complex on platelets. Moreover, the interaction was blocked by anti-GP Ib monoclonal antibodies known to inhibit vWF binding to platelets. The sequence of GP Ib alpha between residues 269-287 has a strong net negative charge due to the presence of 10 glutamic or aspartic acid residues; 5 of these are contained in the sequence of a synthetic peptide (residues 251-279) previously shown to inhibit vWF-platelet interaction. In order to evaluate the possible functional role of these acidic residues, we employed site-directed mutagenesis to express two mutant GP Ib alpha fragments containing asparagine or glutamine instead of aspartic or glutamic acid, respectively. Mutant 1, with substitutions between residues 251-279, failed to bind vWF whether in the presence of ristocetin or botrocetin; in contrast, vWF binding to Mutant 2, with substitutions between residues 280-302, was nearly normal in the presence of ristocetin, but markedly decreased in the presence of botrocetin. Thus, mammalian cells transfected with a truncated cDNA sequence encoding the amino-terminal domain of GP Ib alpha synthesize a fully functional vWF-binding site; acidic residues in the sequence 252-287 are essential for normal function.