Phenotypic heterogeneity in the Gray platelet syndrome extends to the expression of TREM family member, TLT-1.

The Gray platelet syndrome (GPS) is a rare inherited disorder linked to undefined molecular abnormalities that prevent the formation and maturation of alpha-granules. Here, we report studies on two patients from unrelated families that confirm phenotypic heterogeneity in the disease. First we used immunoelectron microscopy (I-EM) to confirm that TREM-like transcript-1 (TLT-1) is mostly localized to alpha-granule membranes of normal platelets. Then we performed Western blotting (WB) and flow cytometry with permeabilized platelets to show that TLT-1 is selectively reduced in the platelets of patient 1, previously noted to be deficient in glycoprotein (GP)VI (Nurden et al., Blood 2004; 104: 107-114). Yet both TLT-1 and GPVI were normally expressed in platelets of patient 2. Usual levels of JAM-C and claudin-5, also members of the immunoglobulin receptor family, were detected in platelets of both patients. In contrast, P-selectin was markedly decreased for patient 1 but not patient 2. Two metalloproteases, MMP-2 and MMP-9 were normally present. As predicted, platelets of patient 1 showed little labelling for TLT-1 in I-EM, whereas residual Fg was seen in small vesicular structures and P-selectin lining vacuoles or channels of what may be elements of the surface-connected canalicular system. Our results identify TLT-1 as a glycoprotein potentially targeted in platelets of GPS patients, while decreases in at least three membrane glycoproteins suggest that an unidentified proteolytic activity may contribute to the phenotype in some patients with this rare disease.

Paradoxical platelet activation was not observed on dissociation of abciximab from GpIIb-IIIa complexes.

The ability of abciximab to bind and dissociate from platelets raises the question of the conformational state of GPIIb-IIIa complexes losing abciximab and the risk of paradoxical drug-induced platelet activation. Platelets incubated with abciximab and mixed in vitro with c7E3 Fab-free platelets lost the drug to the new platelets giving a single platelet population with a unimodal abciximab distribution within 17 h. Prelabeling the receiving platelets with phycoerythrin-labeled anti-GPIb monoclonal antibody (MoAb), permitted their identification by flow cytometry. Binding of PAC-1 and AP6, two MoAbs specific for activated GPIIb-IIIa, was then assessed to both losing and receiving platelet populations during transfer of abciximab. The subpopulation losing c7E3 Fab failed to show increased binding of these MoAbs. However, PAC-1 binding increased in both subpopulations after addition of ADP. Thus GPIIb-IIIa complexes are not in an activated state after dissociation of abciximab unless there is an additional source of activation.

Analysis of the amino acid requirement for a normal alphaIIbbeta3 maturation at alphaIIbGlu324 commonly mutated in Glanzmann thrombasthenia.

Glanzmann thrombasthenia is an inherited bleeding disorder arising from quantitative or qualitative defects of the alphaIIbbeta3 integrin of platelets. Here, we report that PCR-SSCP analysis and DNA sequencing revealed a homozygous single base pair substitution in exon 12 of the IIb gene leading to a Glu(324) (E) to Lys (K) substitution in the alphaIIb subunit in a patient with Type I disease. As this mutation is found on at least 3 continents, the codon for Glu(324) may be a mutational hotspot of the disease. To better understand this mutation, we analyzed the effect of substituting E(324) with A(324), L(324), D(324), Q(324), N(324), S(324), as well as K(324), looking at both alphaIIbbeta3 maturation and cell surface expression in transiently transfected Cos-7 cells. The maturation state of the receptor clearly correlated with the level of cell membrane expression. Maturation efficiency was dependent on the electric charge as well as the size of the side chain of the amino acid present in what is a highly conserved N-terminal position in the third beta-strand of blade 5 of the alphaIIbeta beta-propeller.

A novel hemizygous Bernard-Soulier Syndrome (BSS) mutation in the amino terminal domain of glycoprotein (GP)Ibbeta--platelet characterization and transfection studies.

Glycoprotein (GP) Ib-V-IX is a unique adhesion receptor complex on platelets. Mutations in GPIbalpha, Ibbeta, and IX can lead to the rare bleeding disorder, Bernard-Soulier Syndrome (BSS). Here, we report a novel hemizygous variant of BSS in which Pro29 in one GPIbbeta allele is substituted by a Leu (GPIbbeta:P29L). Fluoresence in situ hybridisation revealed that the 22q11 locus was deleted from the homologous chromosome. The pedigree was determined and revealed inheritance of the GPIbbeta:P29L allele from the father. Flow cytometry with a range of antibodies detected no expression of GPIb-V-IX on the surface of the patient's platelets. Western blotting revealed an absence of GPIbalpha and GPIbbeta from platelet lysates. Co-expression of GPIbbeta:P29L with normal GPIbalpha and GPIX in a heterologous cell system confirmed that the mutant subunit did not support surface expression of the complex. Interestingly, residual expression of GPIbbeta:P29L anchored in the plasma membrane alone was now seen. This novel BSS mutation expressed in heterologous cells is in agreement with recent in vitro evidence that the correct conformation of the amino terminal region of GPIbbeta is required for normal expression of the intact receptor complex.

Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia.

In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibalpha (GPIbalpha). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbalpha N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbalpha, alphaIIb, and beta3 were normally present. Proteins involved in Ca(2+) homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca(2+) ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP(3)-R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal megakaryocytes cultured from peripheral blood CD34(+) cells with TPO. Significantly, megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.

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.

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.

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.

Immunolocalization of P2Y1 and TPalpha receptors in platelets showed a major pool associated with the membranes of alpha -granules and the open canalicular system.

P2Y(1) and thromboxane-prostanoid-alpha (TPalpha) receptors on platelets belong to the G-protein-coupled 7-transmembrane domain family. They transmit signals for shape change, mobilization of calcium, and platelet aggregation. Immunogold labeling with a monoclonal antibody (MoAb) to the amino-terminal domain of P2Y(1) and a polyclonal antibody to the C-terminal domain of TPalpha revealed that while present at the platelet surface, both receptors were abundantly represented inside the platelet. Specifically, receptors were found in membranes of alpha-granules and elements of the open-canalicular system. A similar organization was found in mature megakaryocytes. Activation of platelets by adenosine diphosphate (ADP) and the thromboxane A(2) (TXA(2)) analog, I-BOP [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha)-7-(3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid], increased the labeling of both P2Y(1) and TPalpha at the surface and in intracellular pools, suggesting that activation resulted in greater antibody accessibility to the receptor. A return to a platelet discoid shape and to basal values of labeling accompanied receptor desensitization. Platelets lacking the P2Y(12) ADP receptor normally expressed P2Y(1) and TPalpha, both before and after activation. Studies with the anti-ligand-induced binding site (anti-LIBS) MoAb, AP-6, confirmed that stored fibrinogen associated with internal pools of alpha(IIb)beta(3) at the start of secretion in a microenvironment containing agonist receptors. Pharmacologic antagonism of ADP or TXA(2) receptors in antithrombotic therapy may need to take into account blockade of internal receptor pools.

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome.

We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking alpha-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor gamma-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C gamma 2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the literature.

A novel missense mutation shows that GPIbbeta has a dual role in controlling the processing and stability of the platelet GPIb-IX adhesion receptor.

Glycoprotein (GP) Ibalpha is a major adhesive receptor of platelets, surface expressed as part of the GPIb-IX-V complex. However, important questions about how the four gene products (Ibalpha, Ibbeta, IX, and V) composing this complex are processed remain. A deficiency of or nonfunctioning GPIb-IX-V is characteristic of the Bernard-Soulier syndrome (BSS), an inherited bleeding disease. We now report a BSS variant whose platelets have little or no GIbbeta or GPIX, but where residual GPIbalpha was selectively located in flow cytometry by monoclonal antibodies (WM23 and Bx-1) recognizing denatured epitopes. Whereas WM23 immunoprecipitated GPIbalpha (130 kDa), GPIX, and GPIbbeta from control platelets, a single surface protein of approximately 66 kDa was obtained for the patient. DNA sequencing revealed a homozygous Asn(64) --> Thr substitution in the GPIbbeta from the patient. This substitution modified a conserved residue in the COOH-terminal region flanking the single-copy leucine-rich domain of GPIbbeta. When GPIbbeta64Thr was coexpressed in a stable CHO cell line with wild-type GPIbalpha and GPIX, flow cytometry and confocal microscopy failed to show GPIb-IX complexes at the cell surface. Intracellular GPIbalpha and GPIbbeta were detected and largely confined to the endoplasmic reticulum, and little GPIX was seen. GPIbalpha was immunoprecipitated as a 66-70 kDa protein in (35)S metabolic studies and lacked O-glycosidic side chains. Also, it was not disulfide bound to the mutated GPIbbeta. Thus, a single amino acid substitution in the extracellular domain of GPIbbeta can affect both the maturation of GPIbalpha and GPIX stability. GPIbbeta has a pivotal role in regulating GPIb-IX-V biosynthesis.