Complement component C3 mediates Th1/Th17 polarization in human T-cell activation and cutaneous GVHD.

The complement system has been shown to regulate T-cell activation and alloimmune responses in GVHD. Mice deficient in the central component of complement system C3 have significantly lower GVHD-related mortality/morbidity, and C3 modulates Th1/Th17 polarization in mouse GVHD. To investigate whether anticomplement therapy has any impact on human T-cell activation, a drug candidate Compstatin was used to inhibit C3 activation in this study. We found the frequency of IFN-γ (Th1)-, IL-4 (Th2)-, IL-17 (Th17)-, IL-2- and TNF-α-producing cells were significantly reduced among activated CD4(+) cells in the presence of Compstatin. Compstatin treatment decreased the proliferation of both CD4(+) and CD8(+) T cells upon TCR stimulation. However, Compstatin does not affect the production of IL-2 and TNF-α in activated CD8(+) T cells, and the differentiation of CD8(+) T cells into distinct memory and effector subsets remained intact. Furthermore, we examined complement deposition in skin and lip biopsy samples of patients diagnosed with cutaneous GVHD. C3 deposition was detected in the squamous epithelium and dermis, blood vessels and damaged sweat glands, and was associated with gland damage and regeneration. We conclude that C3 mediates Th1/Th17 polarization in human T-cell activation and skin GVHD in patients.

Abnormal platelet function in C3-deficient mice.

UNLABELLED: SUMMARY BACKGROUND AND OBJECTIVES: The complement system is a biochemical cascade composed of several plasma proteins that can interact with endothelial cells and blood cells, including platelets. In order to investigate the effect of the complement system on platelets, we studied platelet function in C3-deficient mice that lack complement activity. METHOD AND RESULTS: Tail-cut bleeding time was prolonged and platelet aggregation in response to protease-activated receptor-4 (PAR4) peptide was decreased in C3-deficient mice as compared with wild-type littermates. Platelet aggregation in response to other agonists (ADP and collagen) was similar between C3-deficient mice and their normal littermates. Isolated platelets from wild-type mice aggregate less in C3-deficient plasma than in normal plasma, and, conversely, addition of plasma from wild-type mice or plasma-purified C3 improved aggregation of C3-deficient platelets. We also monitored the formation of murine arteriole or venule thrombi in an intravital microscopy thrombosis model. We found that C3-deficient mice had a significantly delayed thrombotic response in arterioles as compared with their wild-type littermates. Furthermore, thrombi in C3-deficient mice were less stable and embolized more frequently than those in wild-type mice. CONCLUSIONS: Platelets of C3-deficient mice have subnormal function, resulting in a prolonged tail-cut bleeding time and delayed thrombosis after vessel wall injury.

Role of Kozak sequence polymorphism of platelet glycoprotein Ibalpha as a risk factor for coronary artery disease and catheter interventions.

OBJECTIVES: We sought to determine the role of the -5T/C polymorphism of the platelet glycoprotein (GP) Ibalpha as a potential risk factor for coronary artery disease (CAD) and adverse events complicating a coronary catheter intervention. BACKGROUND: The platelet GP Ib-IX-V receptor complex plays a crucial role in arterial thrombus formation. The -5T/C polymorphism of GP Ibalpha is associated with increased receptor density. METHODS: We genotyped 1,000 patients with angiographically confirmed CAD, as well as 1,000 age- and gender-matched control subjects, for this polymorphism by polymerase chain reaction/restriction fragment length polymorphism. Among the patients with CAD, 269 underwent percutaneous transluminal coronary angioplasty (PTCA), 103 underwent directional coronary atherectomy and 278 underwent stenting. This intervention group was followed for a 30-day composite end point of target vessel revascularization, myocardial infarction or death. RESULTS: Carriers of the -5C allele were significantly over-represented in the group of patients developing acute coronary syndromes (relative risk [RR] 1.43, 95% confidence interval [CI] 1.05 to 1.95, p = 0.02). The -5C allele furthermore predicted an increased risk for developing complications after PTCA (RR 3.75, 95% CI 1.15 to 12.27, p = 0.029). CONCLUSIONS: The -5C allele of the GP Ibalpha Kozak polymorphism may represent a risk factor in clinical conditions in which thrombosis plays an important role, such as in acute coronary syndromes and in complications after PTCA.

Platelet glycoprotein Ibalpha Kozak polymorphism is associated with an increased risk of ischemic stroke.

Platelets are pivotal to the process of arterial thrombosis resulting in ischemic stroke. Occlusive thrombosis is initiated by the interaction of von Willebrand factor (vWf) and platelet glycoprotein (GP) Ibalpha. Three polymorphisms have been described in GP Ibalpha (Kozak T/C polymorphism, variable number of tandem repeats [VNTR], and the human platelet antigen 2a [HPA-2a] [Thr] or HPA-2b [Met] at position 145), each of which may enhance the vWf and GP Ibalpha interaction. This study investigated whether these polymorphisms are candidate genes for first-ever ischemic stroke. A hospital-based case-control study was conducted of 219 cases of first-ever ischemic stroke and 205 community controls randomly selected from the electoral roll and stratified by age, sex, and postal code. The subtypes of stroke were classified, the prevalence of conventional risk factors was recorded, and blood was collected to perform genotyping analysis for Kozak C or T alleles, VNTR, and HPA-2a/b. It was found that the Kozak T/C genotype was over-represented in the stroke group (32.2%) compared with controls (22.8%) (odds ratio [OR], 1.6; 95% confidence interval [CI], 1.03-2.54; P <.03), and the association was still present even after adjusting for conventional risk factors. There was a trend in the increased prevalence of HPA-2a/b in stroke patients (15%) compared with controls (9.9%) (adjusted OR, 1.8; 95% CI, 0.94-3.4; P =.07). No associations were seen with the VNTR polymorphism or with any of the polymorphisms with stroke subtype. It was concluded that the Kozak T/C polymorphism, which is associated with an increase in platelet GP Ibalpha surface expression, is an independent risk factor for first-ever ischemic stroke.

Human polymorphism of P-selectin glycoprotein ligand 1 attributable to variable numbers of tandem decameric repeats in the mucinlike region.

Platelet GP Ibalpha and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1) are membrane mucins with a number of structural and functional similarities. It was investigated whether, like GP Ibalpha, PSGL-1 is affected by a variable number of tandem repeat polymorphism in its mucin-like region. By polymerase chain reaction amplification of the genomic region encoding the PSGL-1 repeats, 3 allelic variants were identified in the human population. The 3 alleles-A, B, and C-from largest to smallest, contained 16, 15, and 14 decameric repeats, respectively, with the B variant lacking repeat 2 and the C variant retaining repeat 2 but lacking repeats 9 and 10. Allele frequencies were highest for the A variant and lowest for the C variant in the 2 populations studied (frequencies of 0.81, 0.17, and 0.02 in white persons and 0.65, 0.35, and 0.00 in Japanese). Thus, PSGL-1 is highly polymorphic and contains a structural polymorphism that potentially indicates functional variation in the human population.

Bernard-Soulier syndrome in a patient doubly heterozygous for two frameshift mutations in the glycoprotein ib alpha gene.

We report here the genetic basis of Bernard-Soulier syndrome in a compound heterozygote for two mutant glycoprotein (GP) Ib alpha alleles. One allele contained a novel four base-pair deletion (TGAG) that eliminated the last base of the codon for Ser39 (AGT) and the entire codon for Glu40 (GAG), causing a reading frame shift that yielded a stretch of 51 amino acids before a premature stop codon. The other allele also contained a frame-shift mutation, caused by deletion of the last two bases of the codon for Tyr492 (TAT). This allele produced a truncated glycoprotein Ib alpha that, although not expressed on the surface of the patient's platelets, was detectable in the plasma. The second allele has been identified previously by our group and other investigators as the cause of Bernard-Soulier syndrome in patients of northern European ancestry. This allele carried a haplotype identical to those of the previously reported cases, with the following polymorphic markers: two tandem repeats in the VNTR region, C at nucleotide -5 from the ATG start codon and a substitution of G for A in the third base for codon Arg342. These findings suggest that this particular Bernard-Soulier mutation occurred once on the background of a rare haplotype and has spread throughout the northern European population.

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region.

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.

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)

Kozak sequence polymorphism of the glycoprotein (GP) Ibalpha gene is a major determinant of the plasma membrane levels of the platelet GP Ib-IX-V complex.

Despite the known importance of the sequences surrounding ATG start codons (Kozak sequences) for efficient translation of proteins, few reports have appeared that describe the natural variations in these sequences. Here, we report a human polymorphism in the Kozak sequence of the platelet adhesion receptor, glycoprotein (GP) Ibalpha, a component of the GP Ib-IX-V complex, which mediates the initial adhesion of platelets to the blood vessel wall following injury. The polymorphism is based on the presence of either thymine (T) or cytosine (C) at position -5 from the initiator ATG in the GP Ibalpha gene. The less common allele, -5C, represented 8% to 17% of the alleles in four ethnic populations surveyed. This allele more closely resembles the sequence considered optimal for efficient initiation of protein translation and is associated with increased expression of the receptor on the cell membrane, both in transfected cells and in the platelets of individuals carrying the allele. In vitro transcription/translation studies indicate that the increased expression results from more efficient translation of the -5C form of the GP Ibalpha mRNA. Other mutations made to approximate more closely the consensus sequence described by Kozak did not increase expression of the receptor. This is the first known description of Kozak sequence polymorphism as a determinant of the surface levels of a cell adhesion receptor. This polymorphism may influence an individual's susceptibility for the development of cardiovascular disease.

Bernard-Soulier syndrome caused by a dinucleotide deletion and reading frameshift in the region encoding the glycoprotein Ib alpha transmembrane domain.

We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ib alpha was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ib alpha degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ib alpha, GP Ib beta, and GP IX genes. The patient was homozygous for a specific GP Ib alpha allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ib alpha gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T --> C) in the second nucleotide of exon 2, which is in the 5' untranslated region of the GP Ib alpha transcript, and a silent mutation in the third base of the codon for Arg342 (A --> G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ib alpha amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ib alpha cDNA, alone and in combination with the 5' mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ib alpha expression on the cell surface. Expression was not decreased further by addition of the 5' mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ib beta, as demonstrated by their coimmunoprecipitation with a GP Ib beta antibody.

The cytoplasmic domain of glycoprotein (GP) Ibalpha constrains the lateral diffusion of the GP Ib-IX complex and modulates von Willebrand factor binding.

To study the role of the glycoprotein (GP) Ibalpha cytoplasmic domain in the mobility of the GP Ib-IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines expressing GP Ib-IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibalpha or one of a series of GP Ibalpha truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibalpha monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibalpha was negligible. However, when only six amino acids were removed from the GP Ibalpha carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibalpha), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55-67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibalpha cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibalpha fixes the position of the GP Ib-IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.