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Objective: Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ-/-) or the complex was depleted. The development of α2β1-/- and GPVI-/- mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro., Approach and Results: To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1-/-, FcRγ-/-, and GPVI-/- mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ-/- platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI-/- and wild type platelets. The difference between FcRγ-/- and GPVI-/- platelet phosphotyrosine levels correlated with the in vivo thrombosis findings., Conclusion: Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings suggest a more complex signaling network downstream of the platelet collagen receptors than previously appreciated.

Objective and Design: Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at -1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC., Results: IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC., Conclusions: Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.

We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK α2β1 develop normally, are fertile, and like their systemic α2β1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet α2β1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of α2β1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of α2 integrin in selected cell types.

Purpose of Review: This review summarizes our current knowledge of common gene variants (polymorphisms) that have small individual effects on platelet function in humans, but can cumulatively lead to hyperreactive platelets and increase risk for negative outcomes in thrombotic disorders., Recent Findings: Candidate gene association and genome-wide association studies (GWAS) have identified loci that include single nucleotide polymorphisms, which exert a cumulative effect on platelet function by modifying basic platelet parameters, such as mean platelet volume (MPV) or platelet count, by altering the expression or activity of key platelet receptors, or by influencing downstream effector pathways utilized by these receptors., Summary: Variation in MPV between normal individuals is responsible for roughly a two-fold range in platelet protein content, including key surface receptors and reactive granule constituents, the association of ADRA2, GP1BA, GP6, ITGA2 and P2Y12 variants with platelet reactivity, initially identified by candidate gene analyses, has now been validated by genome-wide approaches in much larger individual cohorts, and GWAS have identified novel gene variants, most notably PEAR1, that participate in variation in platelet reactivity among normal individuals, all of which contribute to a genetic basis for differences in platelet reactivty among normal individuals.

A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2β1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles.

Objective: The interindividual variation in platelet α(2)β(1) exceeds a 2-fold variance in platelet α(IIb)β(3) level. Our objective was to parse the contribution of mean platelet volume (MPV) and integrin gene alleles to this variation in large cohorts of patients with acute coronary syndrome (ACS) and normal subjects., Methods and Results: Platelet α(IIb)β(3) and α(2)β(1) levels were measured by flow cytometry in whole blood from 320 ACS patients and 128 normal subjects and compared with MPV, platelet count, ITGA2 rs1126643, and ITGB3 rs5918 alleles. In all subjects, a strong direct correlation was found between MPV and α(IIb)β(3) level (P<0.001). Neither MPV nor α(IIb)β(3) level correlated with ITGB3 rs5918 alleles. In the case of α(2)β(1) level, MPV contributed modestly, whereas ITGA2 rs1126643 exerted a greater effect. An inverse correlation was found between MPV and the rs1126643 minor allele., Conclusions: MPV is the major effector of platelet α(IIb)β(3) level, whereas the ITGA2 rs1126643 alleles influence α(2)β(1) level more than MPV does. The rs1126643 minor allele, associated with lower MPV, likely exerts this effect via the influence of α(2)β(1) on megakaryocyte maturation. Because of the hyperactivity of larger platelets, MPV is an accurate metric of risk for adverse outcome in ACS.

Genetic and environmental factors contribute to a substantial variation in platelet function seen among normal persons. Candidate gene association studies represent a valiant effort to define the genetic component in an era where genetic tools were limited, but the single nucleotide polymorphisms identified in those studies need to be validated by more objective, comprehensive approaches, such as genome-wide association studies (GWASs) of quantitative functional traits in much larger cohorts of more carefully selected normal subjects. During the past year, platelet count and mean platelet volume, which indirectly affect platelet function, were the subjects of GWAS. The majority of the GWAS signals were located to noncoding regions, a consistent outcome of all GWAS to date, suggesting a major role for mechanisms that alter phenotype at the level of transcription or posttranscriptional modifications. Of 15 quantitative trait loci associated with mean platelet volume and platelet count, one located at 12q24 is also a risk locus for coronary artery disease. In most cases, the effect sizes of individual quantitative trait loci are admittedly small, but the results of these studies have led to new insight into regulators of hematopoiesis and megakaryopoiesis that would otherwise be unapparent and difficult to define.

Background: We have identified a cytosine-adenosine (CA) repeat length polymorphism in the 5'-regulatory region of the human integrin alpha2 gene ITGA2 that begins at -605. Our objective was to establish the contribution of this polymorphism to the regulation of integrin alpha2beta1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s)., Methodology/principal Findings: In combination with the SNP C-52T, previously identified by us as a binding site for the transcription factor Sp1, four ITGA2 haplotypes can be distinguished, in the order in which they enhance ITGA2 transcription: (CA)(12)/-52C>(CA)(11)/-52C>(CA)(11)/-52T>(CA)(10)/-52T. By DNA affinity chromatography and chromatin immunoprecipitation (ChIP) assays, we show that poly (ADP-ribose)polymerase-1 (PARP-1) and Ku80/70 bind specifically and with enhanced affinity to the longer (CA)(12) repeat alleles., Conclusions/significance: The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)(12) alleles of ITGA2 coincides with enhanced alpha2beta1 expression. The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2. These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin alpha2beta1 expression in human platelets and other cells.

Background: The Platelet Function Analyzer-100 (PFA-100) is widely used to measure platelet reactivity in whole blood under high shear., Objective: To characterize the genetic component of platelet reactivity among normal individuals, using the PFA-100., Methods: We compared baseline platelet reactivity with sex, age, platelet count, hematocrit, plasma von Willebrand factor antigen (VWF:Ag), and alleles of seven candidate genes: integrin subunits alpha2 (ITGA2) and beta3 (ITGB3), platelet glycoproteins GPIbalpha (GP1BA) and GPVI (GP6), purinogenic receptors (P2RY1 and P2RY12) and cyclooxygenase-1 (COX1)., Results: Based on linear and logistic regression models, we report an inverse correlation between baseline closure time (CT) initiated by collagen plus epinephrine (CEPI) and plasma VWF:Ag level, ITGA2 807T and P2RY1 893C, and an inverse correlation between baseline CT initiated by collagen plus adenosine diphosphate (CADP) and P2RY1 893C or GP1BA -5C., Conclusions: These results indicate that genetic polymorphisms in ITGA2 and P2RY1 combine with plasma VWF:Ag levels to modulate baseline platelet reactivity in response to collagen plus EPI, while genetic differences in P2RY1 and GP1BA significantly effect platelet responses to collagen plus ADP. Our results demonstrate that the PFA-100 can be used to evaluate the effects of genetic predictors of platelet function.

We studied how integrin alpha2beta1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca(2+) concentration ([Ca(2+)](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca(2+)](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca(2+) chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca(2+) chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca(2+) responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca(2+) peaks, whereas those lacking alpha2beta1 showed markedly reduced to absent alpha-like and no gamma-like Ca(2+) peaks. Specific alpha2beta1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha2beta1 may generate Ca(2+) signals that are reinforced by GPVI and required for subsequent longer-lasting Ca(2+) oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha2beta1 in response to collagen stimulation.

The 2 most common haplotypes of human GP6, GP6a and GP6b, generate the allelic isoforms glycoprotein VI (GPVI)a and GPVIb that differ by 5 amino acids: S219P, K237E, and T249A in the ectodomains, and Q317L and H322N in the cytoplasmic domain. By quantitative Western blot, we found no association between GP6 genotype and total platelet GPVI content among 132 normal subjects. When expressed as soluble products or as membrane-associated receptors, GPVIa and GPVIb have identical affinities for type I collagen, collagen-related peptide, or convulxin. However, the cytoplasmic domain substitutions in GPVIb have a significant effect on GPVI-dependent subcellular associations and ligand-induced signal transduction. L317 increases binding to calmodulin, whereas N322 attenuates binding to Fyn/Lyn. Consistent with the latter finding, convulxin-induced Syk phosphorylation is significantly attenuated in Dami cells stably transfected with GPVIb, relative to GPVIa. This represents direct evidence that haplotype-related GPVI functional differences are inherent in the cytoplasmic domain substitutions, whereby GPVIb binds less strongly to Fyn/Lyn and attenuates the rate and extent of Syk phosphorylation. These allelic differences in GP6a and GP6b explain functional differences in the respective isoforms, but the molecular basis for the several-fold range in GPVI levels of human platelets remains to be determined.

We studied the effect of prophylactic aspirin (ASA) ingestion on platelet function in 463 patients with stroke, transient ischemic attack (TIA) or acute coronary disease (ACD), using the Platelet Function Analyzer-100 (PFA-100). We correlated ASA responsiveness with haplotypes of seven candidate genes, selected for their documented role in platelet function, namely, the genes for integrins alpha2beta1and alphaIIbbeta3 (ITGA2, ITGA2B, and ITGB3), platelet glycoproteins Ibalpha and VI (GPIBA and GP6), the purinergic receptor P2Y1 (P2RY1), and prostaglandin H synthase 1 (PTGS1 = COX1). Non-responsiveness to ASA was defined as the failure of prior ASA ingestion to prolong the PFA-100 closure time (CT) when blood was perfused through cartridges coated with collagen plus epinephrine (CEPI-CT). ASA non-responsiveness was observed in 114 of 463 patients (24.6 %), but was not associated with haplotypes of any of the seven candidate genes. There was also no association between any haplotypes and the CT when blood was perfused through cartridges coated with collagen plus ADP (CADP-CT). The ASA non-responsive cohort had significantly increased whole blood platelet counts (p = 0.03) and plasma von Willebrand Factor antigen levels (p < 0.001), which likely contributes to resistance to the inhibitory effects of ASA in the PFA-100.

We report a 6-year-old girl with amegakaryocytic thrombocytopenia, the first case of this rare congenital disorder not to have an MPL gene mutation. Although no mutations were identified in MPL, Mpl protein was absent in the platelets and TPO induced phosphorylation of the Janus tyrosine kinase 2 (Jak2) was not detected. In addition to the defect of Mpl, the patient demonstrated markedly reduced expression of glycoprotein VI (GPVI) in contrast to normal expression of other platelet-specific proteins GPIb alpha, GPIb beta, and GPIIb. To explore the causes for the absence of Mpl, the entire coding region of Jak2 and AML1 were sequenced and no mutations were identified. To our knowledge, this is the first report that describes a case of amegakaryocytic thrombocytopenia that is not caused by a mutation in MPL and demonstrates the severe impairment of GPVI expression on platelets.

Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 x 1/SvJ x C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6(-/-) mice is uniform and is not affected by genetic background. However, the same Gp6(-/-) mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide single-nucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score = 6.9, P < .0001) that we designate Modifier of hemostasis (Mh). Our results indicate that one or more modifier genes in Mh control the extent to which in vivo platelet thrombus formation is disrupted by the absence of platelet GPVI.

The integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2, and the cell cycle regulation gene PELO, embedded within ITGA1 intron 1. ITGA1 contains a CArG box that is bound by serum response factor (SRF), while PELO contains two Sp1 binding elements. A comparison of mRNA levels in megakaryocytic (MK) and non-megakaryocytic (non-MK) cell lines and an analysis of the transcriptional activity of promoter-LUC reporter gene constructs in transfected cells revealed that ITGA1 is selectively suppressed in the MK lineage. Sodium bisulfite genomic sequencing established that a CpG-rich ITGA1 promoter region (-209/+115) is fully methylated at 19 CpG sites in MK cells that do not express alpha1beta1, but completely demethylated in expressing cells. In vitro methylation of ITGA1 suppresses transcription, while treatment of megakaryocytic cells with 5-aza-2'-deoxycytidine, but not Trichostatin A, resulted in de novo expression of ITGA1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, we observed rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of alpha1beta1 regulation that coincides with the initiation of megakaryocyte differentiation.

There is a 2-fold variation in platelet integrin alpha2beta1 levels among inbred mouse strains. Decreased alpha2beta1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet alpha2beta1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L-specific siRNA. Thus, decreased surface alpha2beta1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1.

Background: Convulxin (CVX), a C-type lectin from the venom of Crotalus durissus terrificus, is a potent activator of human platelets, binding predominantly to glycoprotein (GP)VI. Native CVX is an octamer composed of four alphabeta-heterodimers [(alphabeta)(4)]. Two different native sequences have been reported, one bearing lysine (K), the other glutamic acid (E), at beta chain residue 89, but the physiological relevance of this difference is unknown., Objective: We used the Drosophila S2 system to express recombinant CVX (rCVX) heterodimers (alphabeta) and site-directed mutagenesis to evaluate the influence of multimer size and the substitution betaK89E on CVX function., Methods: By flow cytometry, native CVX and both recombinant forms bind to human platelets in whole blood. By surface plasmon resonance (BIAcore, Piscataway, NJ, USA), the calculated equilibrium dissociation constants (K(D)) were: rCVX alphabeta89K, 11.3 x 10(-8) m; rCVX alphabeta89E, 9 x 10(-8) m; and native CVX, 2.8 x 10(-8) m., Results: Thus, the affinities of the two rCVX forms for human, recombinant GPVI are essentially the same, but the relative affinity of native CVX is about 3-fold higher. The minimum concentration of native CVX that induces maximal human platelet aggregation (70 pm) is roughly 400-fold lower than that of either rCVX (29 nm)., Conclusions: These results are consistent with the hypothesis that the ability of the native CVX octamer to cluster mobile GPVI molecules within the platelet membrane may be the single most important factor that contributes to the efficiency with which CVX is able to induce platelet activation.

The extracellular matrix protein, laminin, supports platelet adhesion through binding to integrin alpha6beta1 In the present study, we demonstrate that human laminin, purified from placenta, also stimulates formation of filopodia and lamellipodia in human and mouse platelets through a pathway that is dependent on alpha6beta1 and the collagen receptor GPVI. The integrin alpha6beta1 is essential for adhesion to laminin, as demonstrated using an alpha6-blocking antibody, whereas GPVI is dispensable for this response, as shown using "knockout" mouse platelets. On the other hand, lamellipodia formation on laminin is completely inhibited in the absence of GPVI, although filopodia formation remains and is presumably mediated via alpha6beta1 Lamellipodia and filopodia formation are inhibited in Syk-deficient platelets, demonstrating a key role for the kinase in signaling downstream of GPVI and integrin alpha6beta1 GPVI was confirmed as a receptor for laminin using surface plasmon resonance spectroscopy and by demonstration of lamellipodia formation on laminin in the presence of collagenase. These results identify GPVI as a novel receptor for laminin and support a model in which integrin alpha6beta1 brings laminin to GPVI, which in turn mediates lamellipodia formation. We speculate that laminin contributes to platelet spreading in vivo through a direct interaction with GPVI.

We analyzed the association of bleeding severity with candidate gene haplotypes within pedigrees of 11 index cases of von Willebrand disease (VWD) type 2 (two type 2A, three type 2B and six type 2M), using the QTL Association model (MENDEL 5.5). In addition to the 11 index cases, these pedigrees included 47 affected and 49 unaffected relatives, as defined by VWF mutations and/or phenotype. A bleeding severity score was derived from a detailed history and adjusted for age. Donors were genotyped using a primer extension method, and eight candidate genes were selected for analysis. VWF antigen (or ristocetin cofactor activity) levels had the strongest influence on bleeding severity score. After Bonferroni correction for multiple testing, only ITGA2 promoter haplotype -52T was associated with an increased bleeding severity score (P < 0.01). This association remained statistically significant when the three type 2B pedigrees were excluded (P = 0.012) or when gender-specific bleeding categories were excluded (P < 0.01). The major haplotypes of seven other candidate genes, GP1BA, ITGA2B, ITGB3, GP6, VWF, FGB, and IL6, were not associated with bleeding severity. These results establish that genetic differences in the expression of the integrin subunit alpha2 can influence the bleeding phenotype of VWD type 2 and complement our previous findings in VWD type 1. Genetically controlled attenuation of platelet collagen receptor expression can influence risk for morbidity in clinical settings where hemostasis is compromised.

Using recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine92-Glycine-Serine94 (N92GS94) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRP), and the snake venom C-type lectin convulxin (CVX). In COS-7 cells transiently transfected with GPVI, deglycosylation with peptide-N-glycosidase F (PNGase F; specific for complex N-linked glycans) or tunicamycin decreases the molecular weight of GPVI and reduces transfected COS-7 cell binding to both CRP and CVX. In stably transfected Dami cells, the substitutions N92A or S94A, but not L95H, resulted in a 30% to 40% decrease in adhesion to CVX, but a 90% or greater decrease in adhesion to CRP and a 65% to 70% decrease in adhesion to type I collagen. Treatment with PNGase F, but not Endoglycosidase H (Endo H) (specific for high-mannose N-linked glycans), produced an equivalent decrease in molecular weight. Neither N92A nor S94A affected the expression of GPVI, based on the direct binding of murine anti-human GPVI monoclonal antibody 204-11 to transfected Dami cells. These findings indicate that N-linked glycosylation at N92 in human GPVI is not required for surface expression, but contributes to maximal adhesion to type I collagen, CRP and, to a lesser extent, CVX.

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-binding protein 1 (GATA-1), specificity protein 1 (Sp1), and Friend leukemia integration 1 (Fli-1). In this report, we show that GPVI expression during megakaryocytic differentiation is dependent on cytosine-phosphate-guanosine (CpG) demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI-nonexpressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI-expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+-enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human C-myeloproliferative leukemia virus ligand (c-Mpl), were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation, which can be directly initiated by TPO.

von Willebrand disease (VWD) type 1 is difficult to diagnose because of bleeding variability and low heritability of von Willebrand factor (VWF) levels. We compared a bleeding severity score and bleeding times to candidate gene haplotypes within pedigrees of 14 index cases, using a covariance components model for multivariate traits (Mendel: QTL Association). These pedigrees included 13 affected and 40 unaffected relatives, as defined by plasma ristocetin cofactor (VWF:RCo) levels. The bleeding severity score was derived from a detailed history. Donors were genotyped using a primer extension method, and 9 candidate genes were selected for analysis. VWF:RCo levels had the strongest influence on bleeding severity score and bleeding time. ITGA2 haplotype 2 (807C) and ITGA2B haplotype 1 (Ile(843)) were each associated with increased bleeding severity scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro(219)) was also associated with increased scores (P = .03) after adjustment for donor age. No association was observed with 6 other candidate genes, GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (-1793G; P = .02). These results establish that genetic differences in the adhesion receptor subunits alpha(2), alpha(IIb,) and GPVI can influence the phenotype of VWD type 1.

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.

Monoclonal antibody (mAb) AP7.4 is an anti-integrin antibody recombinantly expressed in Escherichia coli specific to alphavbeta3. It is known that in a variety of RGD-containing molecules, ligand specificity is regulated by structural determinants within the immediate vicinity of the RGD sequence. To better understand the role of the RGD sequence in integrin specificity, we report here the three-dimensional structure of Fab of mAb AP7.4 to a resolution of 2.25 A. The crystals belong to a triclinic space group P1 and the volume of the unit cell is consistent with the presence of two Fab molecules in it. The RGD sequence is located at the tip of a flexible loop in the complementary determining region (CDR-3) of the heavy chain. It has been shown that specific recognition of RGD ligands by their receptors is influenced mainly by the conformation of the tripeptide RGD and the amino acid residues flanking it on either side. Hence, the flexibility of the RGD-carrying loop observed in the crystal structure may stem from the fact that the antibody molecule mimics the function of these cell adhesion molecules.

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collagen-induced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VInull platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VInull or FcR-gammanull animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VInull platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils.

We have tested the DNA of a large series of Glanzmann thrombasthenia patients for polymorphisms in platelet membrane glycoproteins. To our surprise, we noted a high prevalence of the HPA-1b allele of beta3, the minority allele in a normal population. This proved to be due to the presence of nine patients homozygous for the so-called French gypsy mutation (IVS15[ + 1]G-->A) in alphaIIb. Seven of these patients were homozygous for the HPA-1b alloantigen and the other two heterozygous HPA-1a/1b. As the alphaIIb and beta3 genes are both on chromosome 17, it is highly probable that the French gypsy mutation first arose on a chromosome encoding HPA-1b. For other adhesion receptors, no major differences were seen in the distribution of the A1, A2 and A3 alleles in the alpha2 gene, or in the Kozak or HPA-2 polymorphisms of GPIbalpha, suggesting that none of these alleles result in increased survival in Glanzmann thrombasthenia.

Objective: Platelet glycoprotein VI is a collagen receptor belonging to the immunoglobulin-like protein family that is essential for platelet interactions with collagen and is exclusively expressed in the megakaryocytic lineage. The objective of this study was to characterize the human glycoprotein VI gene (GP6) 5' regulatory and promoter regions., Methods and Results: We first used 5' RACE to establish experimentally that the major transcription start site lies 28 bp upstream from the start codon. We next subcloned the 5' regulatory region of GP6 into pGL3-basic [pGL3(-1576)] and used deletion mutagenesis to identify important regulatory regions, comparing the activity of transiently expressed promoter-luciferase constructs in Dami and HeLa cells. We found that megakaryocyte lineage-specific transcription is largely controlled within the segment -191/-39. By site-directed mutagenesis, we confirmed that a GATA-1 site at -176 and an Ets-1 site at -45 play important roles in the regulation of GP6 transcriptional activity., Conclusions: We have determined that the GP6 sequence -191 to -39 represents the core promoter and that transcription is driven largely by GATA-1 (-176) and c-Ets-1 (-45) sites within this segment.

With regard to hemostasis and thrombosis, collagens are among the most important physiologic components of the extracellular matrix in their role as platelet activators. Differences in the rate of platelet activation markedly influence normal hemostasis and the pathological outcome of thrombosis. Thus, collagen receptors, such as the integrin alpha2beta1 and indirectly, GPIb alpha, represent a relatively unexploited target of pharmacological control and are only recently becoming appreciated as potential factors in the generic risk for thrombosis. In general, the importance platelet glycoprotein polymorphisms as genetic risk factors for arterial thrombosis is a new area of human genomics that needs to be carefully addressed. Discrepancies in the degree to which they are reported to contribute to risk for clinical thrombosis will only be resolved once there is a universal standard for clinical study design. Most of the clinical studies differ by patient population size, ethnicity, bias in the selection of patients and controls, plurality in clinical endpoints and variation of environmental factors. Despite these differences, there is substantial evidence that the integrin beta3 PlA2 haplotype, the GPIb alpha Met145 haplotype, the GPIb alpha -5C haplotype and the integrin alpha2 haplotype 1 (807T) each contribute to the risk for and morbidity of thrombotic disease. There may remain dispute as to the extent of their contribution. However, well-designed, large, prospective, genetic and epidemiologic studies are needed to clarify the role of these and other platelet receptor polymorphisms. Most importantly, the cumulative effects of multiple platelet and plasma glycoproteins SNPs to thrombotic risk must be evaluated concurrently. Additional in vitro studies of the functional relevance underlying these polymorphisms are needed to provide a sound biological explanation for the results of clinical correlations. The opportunity now exists to make significant inroads into the development of strategies for the prevention of thrombotic disease.

Context: Collagens are major components of the vascular subendothelium, and the interaction of platelets with collagens initiates normal hemostasis or pathologic arteriothrombosis. Genetic factors that affect the interaction of platelets with collagens could represent risk factors for either arteriothrombosis or excessive hemorrhage. In this regard, we first found that platelet levels of one of the major platelet collagen receptors, integrin alpha(2)beta(1), vary up to 10-fold in normal healthy individuals and that the higher-level phenotype is associated with allele 1 (807T) of the integrin alpha(2) gene. More recently, we found that there is roughly a fivefold range in platelet glycoprotein VI content among normal individuals, which may also influence risk for thromboembolism., Objective: To determine if genetic polymorphisms of platelet glycoproteins involved in collagen-related function are associated with higher risk for thrombotic disorders, such as coronary heart disease, myocardial infarction, or stroke., Methods: We examined the genetic mechanisms responsible for variation in expression levels of the collagen receptor integrin alpha(2)beta(1) and the potential influence of this variation on risk for thrombotic diseases., Results: We found that patients with arteriothrombotic diseases have a higher frequency of alpha(2) allele 1 (associated with higher levels of platelet integrin alpha(2)beta(1)). We further found that platelet glycoprotein VI content directly correlates with platelet prothrombinase activity, suggesting that a higher phenotype of platelet glycoprotein VI also may contribute to increased risk of arteriothrombotic diseases., Conclusion: Genetic polymorphisms that influence the level or function of platelet collagen receptors need to be seriously considered as genetic risk factors for arteriothrombotic diseases.

Extracellular collagens modulate the rate of platelet activation and thereby markedly influence hemostasis and thrombosis. Platelet receptors for collagens, such as the integrin alpha(2)beta(1), platelet glycoprotein (GP) VI or, indirectly, the GPIb complex, are unexploited targets of pharmacological control, and polymorphisms of these receptors have recently become factored into the genetic risk for thrombosis. Seemingly contradictory findings already exist with regard to the contribution of GPIbalpha and integrin alpha(2) polymorphisms, but these discrepancies will be resolved once there is better standardization of clinical studies. There is already substantial evidence that GPIbalpha VNTR A or B alleles, the GPIbalpha-5C allele, and integrin alpha(2) allele 1 (T(807)) each contribute to increased risk for morbidity in thrombotic disease. However, larger, prospective genetic and epidemiological studies are needed to clarify the role of each of these polymorphisms, the contribution of other platelet receptor polymorphisms, and the synergistic effects of combinations of these factors. In addition, in vitro studies that establish the functional relevance of these polymorphisms will provide sound biological explanations for the results of clinical correlation studies.

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Differences in rates of platelet activation induced by extracellular matrix components such as collagens markedly influence normal hemostasis and the pathologic outcome of thrombosis. Thus, platelet collagen receptors, the integrin alpha2beta1, glycoprotein VI, and the glycoprotein Ib complex, represent unexploited targets of pharmacologic control. Polymorphisms of these receptors are now understood as factors that potentially contribute to thrombotic risk. There is substantial evidence that the GPIbalpha variable number of tandem repeats A or B alleles, the -5C allele of GPIbalpha, and the integrin alpha2 allele 1 (T807) each contribute to risk for and morbidity from thrombotic disease. The extent of their individual contributions is disputed. More well-designed, large, prospective, genetic and epidemiologic studies are needed to clarify the role of these and other platelet receptor polymorphisms, and additional in vitro studies are needed to provide a sound biologic explanation for the outcomes of clinical correlations.

Inherited, single-base substitutions are found at only two positions, C(-)52T and C(-)92G, within the proximal 5'-regulatory region (within -1096 to +48) of the human integrin alpha(2) gene. We recently reported that the T(-)52 substitution results in decreased binding of transcription factor Sp1 to adjacent binding sites, decreased transcription of the alpha(2) gene, and reduced densities of platelet alpha(2)beta(1). In this study, we identify an additional Sp1-binding site at position -107 to -99 and show that the adjacent dimorphic sequence C(-)92G also influences the rate of gene transcription. In the erythroleukemia cell line Dami, transfected promoter-luciferase constructs bearing the G(-)92 sequence exhibit roughly a 3-fold decrease in activity relative to the C(-)92 constructs. In transfected CHRF-288-11 megakaryocytic cells, the corresponding activity decreases by 5-fold. DNase I footprinting of the promoter region with Dami nuclear extracts showed a protected segment at -107 to -99 that can be deprotected by coincubation with molar excess of a consensus Sp1 oligonucleotide. Gel mobility shift assays and supershift assays with specific antibodies indicate that Sp1 binds to this region of the alpha(2) gene promoter. Mutation of the Sp1 binding element within -107 to -99 in constructs containing either C(-)92 or G(-)92 abolishes basal promoter activity and eliminates the binding of Sp1. The G(-)92 sequence has a gene frequency of 0.15 in a typical Caucasian population, and the presence of this allele correlates with reduced densities of platelet alpha(2)beta(1). The combined substitution G(-)92/T(-)52 has an additive influence on gene transcription, resulting in an 8-fold decrease in transfected Dami cells or a 20-fold decrease in transfected CHRF-288-11 cells. In summary, the natural dimorphism C(-)92G within the proximal 5'-regulatory region of the human integrin alpha(2) gene contributes to the regulation of integrin alpha(2)beta(1) expression on megakaryocytes and blood platelets and must thereby modulate collagen-related platelet function in vivo.

Genetically controlled variation in alpha2beta1 expression by human blood platelets was previously described. Sixty-two haplotype sequences corresponding to the proximal 5' regulatory region (-1096 to +1) of the alpha2 gene were compared, and a dimorphic sequence -52C>T was identified that is located precisely between 2 tandem Sp1/Sp3 binding elements previously shown to be absolutely required for transcriptional activity of this gene in epithelial cell lines and the erythroleukemic cell line K562. The gene frequency of -52T in a random Caucasian population is approximately 0.35, and the expression of -52T correlates directly with reduced densities of platelet alpha2beta1. In mobility shift analyses, the -52T substitution attenuates complex formation with both Sp1 and Sp3. When transfected into the erythroleukemia cell line Dami, promoter-luciferase constructs bearing the -52T sequence exhibit a 5-fold decrease in activity relative to the -52C construct. In transfected CHRF-288-11 megakaryocytic cells, the corresponding activity decreases by 10-fold. The -52T sequence appears to be in linkage disequilibrium with the previously defined allele A3 (807C; HPA-5b), known to be associated with diminished expression of platelet alpha2beta1. In summary, a natural dimorphism has been identified within the proximal 5' regulatory region of the human integrin alpha2 gene that is responsible for decreased expression levels of the integrin alpha2beta1 on blood platelets through a mechanism that is probably mediated by the nuclear regulatory proteins Sp1 and Sp3.

This review covers new developments and their clinical implications in three areas: platelet antigen polymorphisms, inhibition of platelet glycoprotein IIb-IIIa, and autoimmune thrombocytopenia (ITP). In Section I, Dr. Kunicki reviews platelet polymorphisms and their clinical implications. A current tabulation of the numerous platelet antigens, both those that are platelet specific and not platelet specific, are summarized. The immunogenic clinical implications of these polymorphisms are considered, including fetal and neonatal alloimmune thrombocytopenia, post transfusion purpura, and refractoriness to platelet transfusion. The functional relationship to hemostasis and thrombosis is also discussed, in particular whether one haplotype of the PIA1/PIA2 (HPA-1a/1b) polymorphism predisposes to myocardial infarction. Finally, novel investigations of polymorphisms will be considered, including hormonal induction of certain polymorphisms. In Section II, Dr. Michelson reviews the newest generation of platelet inhibitors, those blocking glycoprotein IIB/IIIA, from the point of view of the hematologist who might be consulted about a patient receiving this form of treatment. The current use of available IIb-IIIa inhibitors and those in trial and the accepted and possible future indications for their use are addressed. The mechanism of action and actual and theoretical advantages and disadvantages of each inhibitor are explored. Scenarios that prompt consultation with a hematologist are presented, including management of bleeding, thrombocytopenia, and management of the patient requiring emergency surgery. In Section III, Dr. Bussel reviews controversies in ITP, looking at both the current state of the art and the potential for the future. Case presentations are used to illustrate the issues in both children and adults. Three primary areas are addressed: 1) the diagnosis of ITP, 2) when and for which patient to recommend splenectomy, and 3) the management of the refractory splenectomized patient who still has a low platelet count and bleeding symptoms.

Platelet adhesion to collagen-coated surfaces in whole blood under flow conditions is mediated by both von Willebrand factor (vWF)-dependent recruitment of the platelet glycoprotein Ib-IX receptor complex and collagen interaction with the integrin alpha2beta1. In type 1 von Willebrand disease (vWD), platelet adhesive functions are impaired due to the decrease in vWF levels in plasma and platelets. There are at least three alleles of the human alpha2 gene, distinguishable by a cluster of silent or noncoding sequence differences within a segment of the gene. Two alleles, associated with low receptor density can be distinguished by nucleotide 807C, while the third allele associated with high receptor density, expresses nucleotide 807T. Gene frequencies of these alleles in a normal population (n = 167) are 0.58 for 807C and 0.42 for 807T. We measured the frequencies of these alleles in symptomatic patients with five types of vWD (type 1, n = 78; type 2A, n = 25, type 2B, n = 14; type 2M, n = 10; and type 3, n = 20). Compared with the normal group, no significant difference in allele frequencies was observed among individuals with types 2A, 2B, 2M, or 3 vWD. However, the frequency of the 807C allele, associated with low collagen receptor density, among type 1 vWD patients (807C =.71; 807T =.29) was significantly higher than that of the normal population (P =.007). Also, in patients with vWD type 1 and borderline to normal ristocetin-cofactor (vWF:RCo) activity values, collagen receptor density correlates inversely with closure time in a high shear stress system (platelet function analyzer [PFA-100]). We propose that low platelet alpha2beta1 density results in less efficient primary platelet adhesion and may result in increased tendency to bleed, as evidenced by the high frequency of this polymorphism in patients with type 1 vWD compared with normal individuals. In addition, this may account for the variability between patients with similar levels of vWF antigen, but strikingly different bleeding histories.

Recently, we have shown that two alleles of the glycoprotein (GP) Ia gene, designated C807 and T807, are associated with low or high platelet GPIa-IIa density and consequently with slower or faster rate of platelet adhesion to type I collagen, respectively. This polymorphism could therefore present a genetic predisposition for the development of thrombotic disease and hemostasis. We investigated the relationship of the GPIa C807T dimorphism to the risk of coronary artery disease (CAD) and myocardial infarction (MI). An allele-specific polymerase chain reaction (PCR) was developed for genotyping of C807T polymorphism. DNA samples from 2237 male patients who underwent coronary angiography on account of coronary heart disease as verified illness or presumptive diagnosis were genotyped. The odds ratio was calculated as an estimate of the relative risk by multiple logistic regression. We found a strong association between the T allele and nonfatal MI among individuals younger than the mean age of 62 years (n = 1,057; odds ratio, 1.57; P =.004). The odds ratio of MI increased for T807 carriers with decreasing age. The highest odds ratio was detected within the youngest 10% of the study sample (<49 years; n = 223; odds ratio, 2. 61; P =.009). In contrast, no evidence of an association between C807T dimorphism with CAD was found. Our findings suggest that inherited platelet GP variations might have an important impact on acute thrombotic disease.

Activation of the platelet integrin alpha(IIb)beta3, an essential step in platelet aggregation, is regulated by intracellular signal pathways (inside-out signaling). In this study, we characterize a 35-year-old Japanese female, HM, with a life-long history of mucocutaneous bleeding. HM showed a Glanzmann thrombasthenia-like phenotype with normal expression of alpha(IIb)beta3, and failure of platelet aggregation induced by various agonists. An activation-independent ligand mimic monoclonal antibody (mAb), OP-G2, and RGDS peptides bound normally to the patient's alpha(IIb)beta3, while an activating anti-beta3 mAb, AP5, induced normal aggregation of HM platelets. The nucleotide sequence of the entire coding region of the patient's alphaIIb and beta3, including the cytoplasmic domains of each subunit, revealed no abnormalities. Agonist-induced phosphorylation of platelet pleckstrin and myosin light chain was not impaired. Recently, we proposed that a Na+/Ca2+ exchanger is involved in inside-out signaling, especially in the case of chymotrypsin-induced alpha(IIb)beta3 activation (Blood 88: 2594, 1996). However, chymotrypsin-induced platelet aggregation occurred normally in patient HM. Measurement of changes in cytosolic free calcium concentration ([Ca2+]i) revealed that the plateau level of [Ca2+]i after thrombin stimulation was significantly inhibited in patient HM. Our data suggest that patient HM exhibits a Glanzmann thrombasthenia-like phenotype associated with an abnormality in inside-out signaling which would otherwise activate alpha(IIb)beta3.

Antibodies are important tools to explore receptor-ligand interactions. The anti-integrin antibody OPG2 binds in an RGD-related manner to the alphaIIb beta3 integrin as a molecular mimic of fibrinogen. The Fab fragment from OPG2 was cocrystallized with a peptide from the beta3 subunit of the integrin representing a site that binds RGD. The crystal structure of the complex was determined at 2.2-A resolution and compared with the unbound Fab. On binding the integrin peptide there were conformational changes in CDR3 of the heavy chain. Also, a significant shift across the intermolecular interface between the CH1-CL domains was observed so that the angle of rotation relating the two domains was reduced by 15 degrees. This unusual conformational adjustment represents the first example of ligand-induced conformational changes in the carboxyl domains of a Fab fragment., (Copyright 1998 Academic Press.)

Three allelic differences in the alpha2 gene are associated with expression levels of the alpha2beta1 integrin on the platelet surface. We have previously defined two linked silent polymorphisms in the alpha2 gene coding region at nucleotides 807 (C or T) and 873 (G or A). We have now identified one rarer nucleotide polymorphism in the coding region at nucleotide 837 (T or C) and four additional linked polymorphisms within the introns that flank these coding sequences. Moreover, we have determined that the alloantigenic Br polymorphism, which resides in a distal coding region at nucleotide 1648, is also linked to the 837 polymorphism. Thus, three alpha2 gene alleles, defined by eight nucleotide polymorphisms, have now been discovered. Allele 1 (807T/837T/873A/Brb) is associated with increased levels of alpha2beta1; allele 2 (807C/837T/873G/Brb) and allele 3 (807C/837C/873G/Bra) are each associated with lower levels of alpha2beta1. Finally, we also show here that the rate of platelet attachment to type I collagen in whole blood under conditions of high shear rate (1,500/s) is proportional to the density of alpha2beta1 receptors on the platelet surface. Thus, the density of platelet alpha2beta1 could have an important impact on platelet adhesion to collagen in whole blood and therefore on platelet function in vivo, contributing to an increased risk of thrombosis or to bleeding in relevant disease states.

The integrin alpha 2 beta 1 is a receptor for collagen that plays a fundamental role in the adhesion of blood platelets to the extracellular matrix. We previously reported that platelet alpha 2 beta 1 levels among randomly selected individuals can vary up to 10-fold and that this correlates with differences in adhesiveness to type-I or type-III collagens. We have now found two linked, allelic polymorphisms within the coding sequence of the alpha 2 gene that correlate with receptor density, TTT/TTC at codon Phe224 and ACA/ACG at codon Thr246. By Southern blot hybridization of specific antisense DNA probes to segments of genomic DNA that encompass each coding region, we have determined the gene frequencies of each allele in a random donor population (n = 65) to be 0.585 (TTC...ACG) and 0.415 (TTT...ACA). There is a statistically significant correlation between the alleles TTT...ACA (codons 224...246) and high receptor density (n = 30; P < .002), whereas the complimentary alleles TTC...ACG are associated with low receptor density. Heterozygous individuals express intermediate levels of this receptor, and familial studies confirm that these allelic polymorphisms are inherited characteristics. These findings prove that the level of platelet alpha 2 beta 1 is an inherited trait. The molecular basis for receptor density remains to be determined, but our findings establish that these silent alleles within the coding sequence of the alpha 2 gene are linked to the genetic basis for variation in receptor density.

The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Fab molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin alphaIIbbeta3. In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin alphaIIbbeta3 versus the vitronectin receptor alphaVbeta3. The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to alphaIIbbeta3 and not at all to any other RGD-cognitive integrin, particularly alphaVbeta3, under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to alphaIIbbeta3 is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to alphaVbeta3, either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to alphaVbeta3 in a cation-dependent manner, but does not bind at all to alphaIIbbeta3 under any conditions. AP7.4 binding to alphaVbeta3 is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to alphaVbeta3. Despite these differences in specificity for alphaIIbbeta3 and alphaVbeta3, AP7 and AP7.4 remain selective for the beta3 integrins and do not bind to cell lines that express the RGD-cognitive integrins alphaVbeta5 or alpha5beta1. These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD motifs can have a profound effect on beta3 integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti-ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

Integrin subunit compatibility within and between species plays a major role in heterodimer assembly and ligand specificity. As an example, human alphaIIb pairs only with human beta3 and does not assemble a heterodimer with beta3 from other species. We use interspecies subunit chimeras to identify molecular requirements for subunit compatibility and show that species-restricted heterodimer assembly depends on a unique hexapeptide VGSDNH in an extended loop of the hypothetical human beta3 MIDAS domain. This allows us to express alphaIIb(1-233) and beta3(111-318) as a soluble, mini-integrin that retains RGD-dependent ligand recognition. Thus, in the case of one integrin, alphaIIbbeta3, the molecular requirements for integrin subunit compatibility and ligand recognition are intimately related.