Generation and rescue of a murine model of platelet dysfunction: the Bernard-Soulier syndrome

The human Bernard-Soulier syndrome is an autosomal recessive disorder of platelet dysfunction presenting with mild thrombocytopenia, circulating "giant" platelets and a bleeding phenotype. The bleeding in patients with the Bernard-Soulier syndrome is disproportionately more severe than suggested by the reduced platelet count and is explained by a defect in primary hemostasis owing to the absence of the platelet glycoprotein (GP) Ib-IX-V membrane receptor. However, the molecular basis for the giant platelet phenotype and thrombocytopenia have remained unresolved but assumed to be linked to an absent receptor complex. We have disrupted the gene encoding the alpha-subunit of mouse GP Ib-IX-V (GP Ibalpha) and describe a murine model recapitulating the hallmark characteristics of the human Bernard-Soulier syndrome. The results demonstrate a direct link between expression of a GP Ib-IX-V complex and normal megakaryocytopoiesis and platelet morphogenesis. Moreover, using transgenic technology the murine Bernard-Soulier phenotype was rescued by expression of a human GP Ibalpha subunit on the surface of circulating mouse platelets. Thus, an in vivo model is defined for analysis of the human GP Ib-IX-V receptor and its role in the processes performed exclusively by megakaryocytes and platelets.     

Nonsense mutation in the glycoprotein Ib alpha coding sequence associated with Bernard-Soulier syndrome.

Three distinct gene products, the alpha and beta chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. We have analyzed the molecular basis of this disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ib alpha but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ib alpha, which by lacking half of the sequence on the carboxyl-terminal side, including the trans-membrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ib alpha allele. Nonsense mutation and truncated GP Ib alpha polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ib alpha alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome.     

Platelet glycoprotein Ib alpha supports experimental lung metastasis

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the alpha-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.     

Increased thrombin responsiveness in platelets from mice lacking glycoprotein V

A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V -/- platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard-Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover, in vitro analysis demonstrated that GP V -/- platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V -/- mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors.     

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb–IIIa in a patient with an unusual platelet membrane glycoprotein composition

The subject (E.B.) is a 63-year-old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb-IIIa complex (AP2, P2). The patient's platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patient's serum inhibited ADP-induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patient's platelets. Levels of GP Ib-IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb-IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ibα in addition to the band in the normal position of GP Ibα. Finally, ADP-stimulated (E.B.) platelets failed to express activation-dependent epitopes on GP IIb-IIIa as recognized by PAC-1, AP6, or F26 and additionally gave a reduced P-selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex-dependent determinant on GP IIb–IIIa and inhibitory of platelet aggregation. Am. J. Hematol. 57:164–175, 1998. © 1998 Wiley-Liss, Inc.     

Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane

Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.     

Amelioration of the macrothrombocytopenia associated with the murine Bernard-Soulier syndrome

An absent platelet glycoprotein (GP) Ib-IX receptor results in the Bernard-Soulier syndrome and is characterized by severe bleeding and the laboratory presentation of macrothrombocytopenia. Although the macrothrombocytopenic phenotype is directly linked to an absent GP Ib-IX complex, the disrupted molecular mechanisms that produce the macrothrombocytopenia are unknown. We have utilized a mouse model of the Bernard-Soulier syndrome to engineer platelets expressing an alpha-subunit of GP Ib (GP Ibalpha) in which most of the extracytoplasmic sequence has been replaced by an isolated domain of the alpha-subunit of the human interleukin-4 receptor (IL-4Ralpha). The IL-4Ralpha/GP Ibalpha fusion is membrane expressed in Chinese hamster ovary (CHO) cells, and its expression is facilitated by the presence of human GP IX and the beta-subunit of GP Ib. Transgenic animals expressing a chimeric receptor were generated and bred into the murine Bernard-Soulier syndrome-producing animals devoid of mouse GP Ibalpha but expressing the IL-4Ralpha/GP Ibalpha fusion sequence. The characterization of these mice revealed a 2-fold increase in circulating platelet count and a 50% reduction in platelet size when compared with platelets from the mouse model of the Bernard-Soulier syndrome. Immunoprecipitation confirmed that the IL-4Ralpha/GP Ibalpha subunit interacts with filamin-1 and 14-3-3zeta, known binding proteins to the GP Ibalpha cytoplasmic tail. Mice expressing the chimeric receptor retain a severe bleeding phenotype, confirming a critical role for the GP Ibalpha extracytoplasmic domain in hemostasis. These results provide in vivo insights into the structural elements of the GP Ibalpha subunit that contribute to normal megakaryocyte maturation and thrombopoiesis.     

Bernard-Soulier syndrome due to the homozygous Asn-45Ser mutation in GPIX: an unexpected, frequent finding in Germany

Bernard-Soulier syndrome (BSS) is a rare inherited disorder with giant platelets, thrombocytopenia and a prolonged bleeding time. These abnormalities are caused by genetic defects of the glycoprotein (GP) Ib-IX complex that constitutes the von Willebrand factor receptor on the platelet surface. Here, we describe four unrelated German patients with low platelet counts and mild bleeding tendency. Three patients had been diagnosed with immune thrombocytopenia (ITP) and were treated with steroids without response. Another patient presented with easy bruising. Peripheral blood smears showed giant platelets. Ristocetin-induced platelet aggregation was almost absent, and quantitative flow cytometry and Western blotting disclosed a greatly reduced surface expression of GPIb-IX. Unexpectedly, sequencing the entire coding regions of GPIbalpha, GPIbbeta and GPIX revealed that all four unrelated patients were homozygous for an A to G mutation in position 1826 of the GPIX gene, constituting a Asn-45Ser change. This mutation has been described before and now represents by far the most often identified molecular defect causing BSS in Caucasians. Because BSS patients are likely to be misdiagnosed with ITP, treatment-resistant ITP patients should be re-evaluated thoroughly. Asn-45Ser genotyping may be a helpful tool for differential diagnosis.     

Flow cytometric analysis of platelets from childrenwith the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP-induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.     

Inhibition of Binding of von Willebrand Factor to the Platelet Glycoprotein Ib-IX Complex, Heparin and Sulfatides by Polyanionic Compounds. The Mechanism of Modulation of the Adhesive Function of von Willebrand Factor

The interaction of the multimeric glycoprotein von Willebrand Factor (vWF) with its platelet membrane receptor, the glycoprotein (GP) Ib-IX complex plays a key role in the initial adhesion of platelets to the vascular subendothelium at high shear blood flow. The GP Ib-IX-binding site is only expressed following activation of vWF, a process that regulates vWF-mediated platelet adhesion. Binding of vWF to the GP Ib-IX complex involves the vWF A1 internal repeat domain, which also contains distinct binding sites for sulfatides, heparin, and the non-physiological modulators of the vWF-GP Ib-IX interaction, ristocetin and botrocetin. With the ultimate aim of further defining the mechanism of vWF modulation, we have analyzed the ability of various polyanionic compounds, including aurintricarboxylic acid, Evans blue, fucoidan, and a range of sulfated and phosphorylated sugars, to inhibit specific binding of purified vWF to immobilized sulfatides and heparin, and the ristocetin- and botrocetindependent binding of vWF to the platelet GP Ib-IX complex. Firstly, it was confirmed using a solid-phase binding assay that, like sulfatides, heparin specifically bound to a purified 39/WkiloDalton fragment of vWF (Leu-480 to Gly-718) that encompasses the A1 domain. Secondly, the ability of a number of polyanionic compounds to inhibit binding of vWF to heparin, but not to immobilized sulfatides, supported previous data suggesting that heparin and sulfatides bind to distinct sites on vWF. In addition, aurintricarboxylic acid, Evans blue and fucoidan all inhibited binding of vWF to both heparin and sulfatides with similar ICso values. Thirdly, many of the compounds tested that inhibited binding of vWF to heparin also effectively inhibited both ristocetin- and botrocetin-dependent binding of vWF to the GP Ib-IX complex on platelets, whereas none of the compounds tested blocked vWF binding to sulfatides and GP Ib-IX but not heparin. The majority of compounds tested inhibited the vWF-platelet interaction to a comparable degree in the presence of ristocetin or botrocetin, suggesting a similar mechanism for inhibition irrespective of the modulator used. These combined experiments provide evidence for an electrostatic model of vWF modulation, and suggest that the heparin-binding domain of vWF may be an important regulatory site involved in the adhesion of vWF to the platelet GP Ib-IX complex.     

Studies on the megakaryocytes of a patient with the Bernard-Soulier syndrome

We have used monoclonal antibodies AP-1 (anti-GP Ib alpha). AP-2 (anti-GP IIb-IIIa) and FMC 25 (anti-GP IX) in immunofluorescence and immunocytochemical studies on megakaryocytes (MK) isolated from a Bernard-Soulier syndrome (BSS) patient whose giant platelets were characteristically deficient in GP Ib-IX complexes. Electron microscopy showed that the patient's MK were similar in size to normal MK. However, a striking feature was the variable and intermittent nature of the demarcation membrane system which was often vacuolar in appearance. Permeabilized mature MK from normal individuals were strongly positive with AP-2, AP-1 and FMC 25. Those from the BSS patient were normal for AP-2, negative for AP-1 but weakly positive with FMC 25. Binding of the monoclonal antibodies to the patient's platelets was evaluated using flow cytometry. The results confirmed the absence of GP Ib alpha from the surface membranes, but showed the presence of small amounts of GP IX distributed throughout the platelet population. Our findings confirm that the membrane lesion in BSS is also to be found in MK and further show that the defect may affect differently individual constituents of the GP-Ib-IX complex.     

Platelet antibodies in immune thrombocytopenia.

Several tests to detect antibodies to platelets in patients with immune thrombocytopenia have been developed over the 40 years since it was first noted that this disorder was mediated by antiplatelet factors. Early tests were crude and not reproducible. A major landmark was the quantitation of IgG immunoglobulin on platelet membrane and the observation by several workers that marked increases of all classes of immunoglobulin occurred on platelets in patients with immune thrombocytopenia. Although at one time accepted as a diagnostic characteristic of autoimmune thrombocytopenia (AITP), the initial euphoria was tempered over the last decade by the realisation that elevation of platelet associated immunoproteins was not pathognomonic of this disorder and that raised levels were seen in several other disease processes, sometimes even when platelet counts were normal. The nature of these immunoproteins needs careful understanding. True platelet autoantibodies will manifest as increased platelet immunoproteins but not all such platelet proteins are platelet antibodies. There is speculation about the existence and the mode of activity of IgA and IgM immunoglobulins, both commonly found on platelets in AITP. It is sometimes almost inconceivable that the extremely large amounts of PAIgG could possibly represent true autoantibody. Immune complexes are found in plenty in these and other disorders in which thrombocytopenia manifest. In such situations it is likely that 'amplified' immune complexes are bound by Fc receptors, as may be found in viral mediated ITP or in septicaemic states. There is now sound evidence that several glycoprotein such as GP IIb/IIIa, GP Ib, GP V, found on platelet membrane, act as target antigen sites for the attachment of antibodies to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Interaction of von Willebrand Factor and the Platelet Glycoprotein Ib-IX Complex

The interaction between blood platelets and the vessel wall represents the initial event in the haemostatic response to vessel injury. At a molecular level, this interaction involves the platelet membrane glycoprotein (GP) Ib-IX complex, von Willebrand factor (vWF) and a component (s) of the subendothelial matrix. In recent years, the primary sequences of both the vWF molecule and the three chains comprising the GP Ib-IX complex, i.e., GP Ib_α, GP Ib_β and GP IX, have been determined, providing further insight into the structure-function relationships of these molecules. As a result, much recent investigation has been directed towards identifying small amino acid sequences in the primary chains of both GP Ib_α and vWF which define the adhesive interaction. This review summarises current data concerning structure-function analysis of vWF and the GP Ib-IX complex and the putative identification of ligand- and receptor-binding domains involved in the interaction of vWF with the GP Ib-IX complex.     

Single amino acid substitution in human platelet glycoprotein Ibbeta is responsible for the formation of the platelet-specific alloantigen Iy(a)

We recently described a new low-frequency platelet alloantigen on the human platelet glycoprotein (GP) Ib-IX complex, termed Iy(a), which was implicated in a severe case of neonatal alloimmune thrombocytopenia. Immunoprecipitation studies with trypsin-treated platelets indicated that the Iy(a) alloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ibalpha (GP Ibalpha(r)) together with GP Ibbeta and GP IX. To elucidate the molecular basis underlying the Iy(a) alloantigen, we amplified GPIbalpha(r), GPIbbeta, and GPIX genes by polymerase chain reaction (PCR). Nucleotide-sequence analysis of these 3 genes showed a G to A transition at position 141 on GPIbbeta gene in a subject positive for Iy(a). This transition resulted in a Gly(15)Glu dimorphism on the N-terminal domain of GPIbbeta. This finding was confirmed by genotyping analysis of 6 Iy(a)-positive subjects by restriction fragment length polymorphism (RFLP) studies using NarI endonuclease. In 300 randomly selected healthy blood donors, one Iy(a)-positive individual was found. Phenotypes determined by monoclonal antibody-specific immobilization of platelet antigens assay and genotypes determined by RFLP were identical in this population. Analysis of Iy(a)-positive platelets showed that the point mutation affected neither the degree of surface expression nor the function of the GP Ibalpha-GP Ibbeta-IX complex on the platelet surface. Transient expression of the GP Ib-IX complex in CHO cells using wild-type GP Ibbeta (Gly(15)) or mutant GP Ibbeta (Glu(15)) allowed us to demonstrate that this single amino acid substitution is sufficient to induce Iy(a) epitope(s). (Blood. 2000;95:1849-1855)     

Defective adhesion of blood platelets to vascular microfibrils in the Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) platelets, which lack the membrane glycoprotein complex Ib-IX, do not adhere to subendothelium. The adhesion of platelets from two patients with BSS to subendothelial microfibrils (MFs) and type I collagen was compared in an in vitro assay adapted to patients with low platelet count. With both patients, platelet adhesion to MFs was strongly defective, whereas the adhesion to collagen was normal. The involvement of GPIb in the MFs-induced platelet adhesion was confirmed by the inhibitory effect of a MoAb (AN51) to the von Willebrand (vWF) factor binding domain of GPIb. The adhesion of platelets to MFs thus requires GPIb-IX and an axis MFs-vWF- GPIb-IX seems therefore to be prevalent in the reactivity of platelets with subendothelium.     

Bernard-Soulier syndrome: quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements

Abstract: Platelets and megakaryocytes have been characterized in a Bernard-Soulier syndrome (BSS) kindred with respect to glycoprotein (GP) membrane receptors and measurements of thrombocytopoiesis. The index patient exhibited lifelong bleeding tendency, moderate thrombocytopenia (35 × 10⁹/1), giant platelets (mean platelet volume 12.5 μm³ compared to 7.5 ± 1.5 μm³ in normals), absent ristocetin-induced platelet agglutination and absent binding of von Willebrand factor (vWF). Flow-cytometric analysis revealed absent platelet binding (0–2%) of monoclonal antibodies (mAb, LJ-P3, LJ-Ibl and LJ-Ibl0) directed against distinct epitopes on membrane GPIbα of the GPIb-IX complex, and normal binding of LJ-P4 mAb directed against GPIIb/IIIa complex (relative to increased platelet surface area). Marrow megakaryocytes also failed to express GPIb-IX complex, but demonstrated normal expression of GPIIb/IIIa. Among 6 asymptomatic family members, the patient's mother and 2 of his 4 children exhibited approximately 50% binding of anti-GPIbα mAb to their platelets by both flow cytometry and direct binding studies using ¹²⁵I-vWF, ¹²⁵I-LJ-Ibl and ¹²⁵I-LJ-Ibl0 mAb. Marrow megakaryocytes were increased in the average cell volume and cytoplasmic granularity with a corresponding increase in ploidy (46% > 16N compared to 22 ± 5% in normal individuals), a pattern typical of megakaryocytes stimulated by thrombocytopenia. Autologous ¹¹¹In-platelet life span was shortened to 4.1 days (compared with 9.5 ± 0.5 days in normal subjects), and the turnover of platelet mass in the circulation was near normal. The data directly demonstrate that the platelet membrane GPIb-IX defect in BSS originates in megakaryocytes at all levels of cell maturation, and exclude the possibility that the receptor abnormality is acquired during cell maturation or after platelets are released into the circulation. Since marrow megakaryocytes exhibited cellular changes consistent with stimulated megakaryocytopoiesis, these results also suggest that thrombocytopenia in this kindred of BSS is a consequence of both decreased platelet survival and ineffective platelet production.     

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.     

Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets

Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin-stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) immunogold staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time. For alpha-thrombin, but not the other agonists, flow cytometry confirmed a dose- and time-dependent decrease in the binding of MoAbs specific for GP Ib alpha (AP-1, Bx-1), GP IX (FMC 25), or to the complex itself (SZ 1). Immunoglold staining performed using standard transmission or scanning electron microscopy high-lighted surface areas devoid of bound antibody. However, a quantitatively normal immunofluorescence was restored if paraformaldehyde-fixed, thrombin-stimulated platelets were permeabilized with Triton X-100 (Sigma Chemical Co, St Louis, MO) before MoAb addition, while immunogold staining was now seen to be concentrated within the interior of the platelet. Glutaraldehyde-fixed samples were then embedded in the resin Lowicryl K4M (Taab Laboratories Equipment Ltd, Aldermaston, England) and immunogold staining performed on thin sections using a polyclonal antibody to glycocalicin. An increased presence of GP Ib-IX complexes within surface-connected membrane systems of the thrombin-stimulated platelets was confirmed. Interestingly, GP Ib-IX movement was opposite to the thrombin-induced externalization of internal pools of GP IIb-IIIa complexes and of the alpha-granule membrane GP, GMP-140.