A novel 196Leu to Pro substitution in the beta3 subunit of the alphaIIbbeta3 integrin in a patient with a variant form of Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited disorder where an absence of platelet aggregation is associated with quantitative or qualitative abnormalities of the alphaIIbbeta3 integrin. In rare patients, amino acid substitutions have provided information on the functional significance of specific domains within alphaIIb or beta3. We now report an elderly male GT patient (R.M.) from the south west of France whose platelets possess a small residual expression of alphaIIbbeta3. Furthermore, the integrin failed to undergo the necessary conformational changes following platelet activation to permit the binding of fibrinogen or activation-dependent monoclonal antibodies despite the presence of an RGD-binding site. Screening of the alphaIIb and beta3 genes by PCR-SSCP revealed a heterozygous mutation at position 685 in exon 5 of the beta3 gene leading to a 196Leu to Pro substitution. 196Leu is a highly conserved amino acid of beta3. The other beta3 allele appeared to be silent. This mutation, inherited from his mother and present in other family members with intermediate levels of alphaIIbbeta3, was close to the MIDAS-like domain of beta3, a fact that appears to explain its effect on alphaIIbbeta3 activation and fibrinogen binding.     

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)     

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.     

AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin alphaIIbbeta3. The N-terminal domain of the alphaIIb subunit is folded in a beta-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of beta3. Analysing the mutations of Italian GT patients we found that a patient had a alphaIIb G236E missense substitution that substitutes a glycine from the highly conserved PhiPhiGPhi motif of blade 4 of the beta-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated alphaIIb in conjunction with normal beta3. Using flow cytometry analysis we found the percentage of HEK cells transfected with alphaIIbG236Ebeta3 that reacted with anti alphaIIbbeta3 was very low. In HEK cells transfected with either alphaIIbbeta3 or alphaIIbG236Ebeta3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against alphaIIb was present in both lysates, although less intense in cells transfected with alphaIIbG236Ebeta3. In reducing condition alphaIIb from cells transfected with alphaIIbbeta3 was nearly all mature, while in cells transfected with alphaIIbG236Ebeta3 the ratio pro-alphaIIb: alphaIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against alphaIIb and immunoblotted with an antibody reacting with beta3. While in immunoblots from cells transfected with alphaIIbbeta3 a band corresponding to beta3 was strongly detectable, in immunoblots originating from cells transfected with alphaIIbG236Ebeta3 no band at the same level of normal beta3 was detected. Immunofluorescence studies showed accumulation of alphaIIbG236Ebeta3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the alphaIIbG236E mutation causes GT by impairing the association with beta3 during biogenesis of the receptor.     

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.     

A Ser752-->Pro substitution in the cytoplasmic domain of beta3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the alphaIIbbeta3 integrin and the platelet granule pool of fibrinogen

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.     

Two novel mutations in the alpha IIb calcium-binding domains identify hydrophobic regions essential for alpha IIbbeta 3 biogenesis

The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.     

Mutations in the beta3 gene giving rise to type I Glanzmann thrombasthenia in two families in Portugal

Glazzmann thrombasthenia is an inherited bleeding syndrome in which an absence of platelet aggregation is associated with quantitative or qualitative deficiencies of the alphaIIbbeta3 integrin. We now describe biochemical and molecular studies on two Portuguese families where platelets lack both surface and intracellular pools of alphaIIbbeta3. DNA extraction was followed by PCR-SSCP analysis of all exons and intronic boundaries in the alphaIIb and beta3 genes. Migration abnormalities were found for PCR fragments encompassing exon 12 (family 1) and exon 10 (family 2). For patient 1, there was a homozygous G to T transition at position 1846 which resulted in a stop codon at codon 616 in the beta3 gene. For patient 2, direct sequencing revealed a homozygous 1347C insert which led to a stop codon at codon 444 in the beta3 gene. For both patients a single mutated allele was inherited from each parent. Evidence is accumulating that nonsense mutations leading to a truncated beta3 may be a frequent cause of type I Glanzmann thrombasthenia in the Iberian peninsula.     

Species differences in small molecule binding to alpha IIb beta 3 are the result of sequence differences in 2 loops of the alpha IIb beta propeller

Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat alphaIIbbeta3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the alphaIIb beta propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human alphaIIbbeta3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the alphaIIbbeta3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with alphaIIb in a different manner than with these small molecules. None of these species-based substitutions affected the ability of alphaIIbbeta3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the alphaIIb beta-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.     

Recombinant factor VIIa restores aggregation of alphaIIbbeta3-deficient platelets via tissue factor-independent fibrin generation

Recombinant factor VIIa (rFVIIa) is a safe and effective prohemostatic drug for patients with Glanzmann thrombasthenia (GT). However, the mechanism of action of rFVIIa in these patients is still unclear. Although patients with GT are characterized by a complete absence of platelet aggregation to a variety of agonists, it has been shown that GT platelets are able to form aggregates, provided polymerizing fibrin is present. We studied the effect of rFVIIa-mediated fibrin formation on aggregation of alphaIIbbeta3-deficient platelets. When washed platelets from GT patients or platelets from healthy volunteers treated with an arginyl-glycyl-aspartyl-containing peptide were activated with collagen in the presence of rFVIIa and purified coagulation factors X, II, and fibrinogen, complete aggregation occurred after a lag phase. Fibrin generation proceeded via rFVIIa-mediated thrombin generation on the activated platelet surface independently of tissue factor. Electron microscopic analysis of alphaIIbbeta3-independent platelet aggregates showed a densely packed structure suggestive of a true platelet-fibrin interaction and not via trapping of platelets into a fibrin network. Also, rFVIIa-mediated alphaIIbbeta3-independent aggregation was demonstrated under conditions of flow using a collagen-coated surface. In conclusion, the efficacy of rFVIIa in GT patients might be explained by induction of alphaIIbbeta3-independent platelet aggregation, which compensates the lack of alphaIIbbeta3-dependent aggregation.     

Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen

Small-molecule alphaIIbbeta3 antagonists competitively block ligand binding by spanning between the D224 in alphaIIb and the MIDAS metal ion in beta3. They variably induce conformational changes in the receptor, which may have undesirable consequences. To identify alphaIIbbeta3 antagonists with novel structures, we tested 33 264 small molecules for their ability to inhibit the adhesion of washed platelets to immobilized fibrinogen at 16 muM. A total of 102 compounds demonstrated 50% or more inhibition, and one of these (compound 1, 265 g/mol) inhibited ADP-induced platelet aggregation (IC(50): 13+/- 5 muM), the binding of soluble fibrinogen to platelets induced by mAb AP5, and the binding of soluble fibrinogen and a cyclic RGD peptide to purified alphaIIbbeta3. Compound 1 did not affect the function of GPIb, alpha2beta1, or the other beta3 family receptor alphaVbeta3. Molecular docking simulations suggest that compound 1 interacts with alphaIIb but not beta3. Compound 1 induced partial exposure of an alphaIIb ligand-induced binding site (LIBS), but did not induce exposure of 2 beta3 LIBS. Transient exposure of purified alphaIIbbeta3 to eptifibatide, but not compound 1, enhanced fibrinogen binding ("priming"). Compound 1 provides a prototype for small molecule selective inhibition of alphaIIbbeta3, without receptor priming, via targeting alphaIIb.     

Upregulation of osteoclast alpha2beta1 integrin compensates for lack of alphavbeta3 vitronectin receptor in Iraqi-Jewish-type Glanzmann thrombasthenia

Osteoclasts utilize alphavbeta3 integrin adhesion to bone matrix during bone resorption. We have generated osteoclasts from the peripheral blood of Iraqi-Jewish patients with Glanzmann thrombasthenia (GT) who are completely deficient in beta3 integrin and exhibit a haemorrhagic diathesis resulting from the absence of platelet alphaIIbbeta3. We show that, in contrast to osteoclasts generated from normal subjects or patients with alphaIIb integrin deficiency, GT osteoclasts lack alphavbeta3. These osteoclasts exhibited a two- to fourfold increase in alpha2 and beta1 integrin expression, whereas other alphav integrins, including alphavbeta5, were not significantly affected. An accompanying decrease in bone resorption was observed, with 44% and 59% declines in pit number and depth, respectively, and resorption lacunae showed abnormal morphology on scanning electron microscopy. However, osteoclasts from GT developed in similar numbers to controls and exhibited an otherwise 'normal' phenotype. We conclude that the observed rise in alpha2beta1 expression compensates for the chronic genetic deficiency of alphavbeta3 in osteoclasts from patients with GT and is sufficient to enable bone resorption to proceed, albeit to a submaximal extent. This explains why Iraqi-Jewish patients with GT do not have osteopetrosis.     

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.     

Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif

The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets.     

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation

Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.     

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

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

Inherited disorders of platelets: an update

PURPOSE OF REVIEW: To overview inherited syndromes that affect platelets and to discuss current data on the molecular origin and management of these rare diseases. RECENT FINDINGS: An increasing number of genes responsible for inherited thrombocytopenias have been identified and these now extend to glycosylation defects. Although Glanzmann thrombasthenia remains the predominant disorder of platelet function, knowledge is increasing of pathologies concerning primary receptors for adhesion and signalling, the activation and secretory pathways, and even the development of procoagulant activity. SUMMARY: These syndromes affect cell adhesion, cell activation, and cell-to-cell contact interactions fundamental in cell biology. Studies on the pathophysiology of alphaIIbbeta3 in platelets have helped unravel the molecular mechanisms of integrin function, and the information gained has resulted in improved antithrombotic therapy. The establishment of national registries and the use of state-of-the-art genomic and proteomic technologies will accelerate progress and help to define how mutations affecting a much larger range of proteins contribute alone or in combination to defining specific platelet phenotypes.     

A push-pull mechanism for regulating integrin function

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.     

The beta3 subunit of the integrin alphaIIbbeta3 regulates alphaIIb-mediated outside-in signaling

Bidirectional signaling is an essential feature of alphaIIbbeta3 function. The alphaIIb cytoplasmic domain negatively regulates beta3-mediated inside-out signaling, but little is known about the regulation of alphaIIb-mediated outside-in signaling. We show that alphaIIb-mediated outside-in signaling is enhanced in platelets of a patient lacking the terminal 39 residues of the beta3 cytoplasmic tail. This enhanced signaling was detected as thromboxane A(2) (TxA(2)) production and granule secretion, and required ligand cross-linking of alphaIIbbeta3 and platelet aggregation. This outside-in signaling was specifically inhibited by a palmitoylated version of a beta3 peptide corresponding to cytoplasmic domain residues R724-R734. Unlike the palmitoylated peptide, the nonpalmitoylated beta3 peptide could not cross the platelet membrane and did not inhibit this outside-in signaling. The physiologic relevance of this beta3-mediated negative regulation of alphaIIb outside-in signaling was demonstrated in normal platelets treated with the palmitoylated peptide and a physiologic agonist. Binding of alphaIIbbeta3 complexes to immobilized peptides demonstrated that a peptide corresponding to beta3 residues R724-R734 appears to bind to an alphaIIb cytoplasmic domain peptide containing residues K989-D1002, but not to control peptides. These results demonstrate that alphaIIb-mediated outside-in signaling resulting in TxA(2) production and granule secretion is negatively regulated by a sequence of residues in the membrane distal beta3 cytoplasmic domain sequence RKEFAKFEEER.