Genetics of platelet reactivity in normal, healthy individuals.

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.

Platelet function defects.

Inherited defects of platelet function are a heterogeneous group of disorders that can result in bleeding symptoms ranging from mild bruising to severe mucocutaneous haemorrhage. These defects may be classified according to their effect on the various steps of platelet microthrombi formation including initiation, extension and cohesion, or based on their particular structural or functional deficiency. Platelet membrane receptor deficiencies result in the rare, but well-characterized syndromes of defective clot initiation, such as Bernard-Soulier Syndrome. Platelet storage pool defects are the most common disorders affecting the extension phase of clot formation. Glanzmann thrombasthenia, with absent or dysfunctional alpha IIb beta 3 receptor is the prototypical defect of the cohesion/aggregation phase of microthrombi formation. Many of these disorders share common treatments although some therapies will have greater efficacy for one patient than another and should be individualized so as to provide optimal control of symptoms. Currently much effort is being put into methods to more rapidly and accurately diagnose patients with platelet disorders and to initiate appropriate therapy and prevent life threatening bleeding.

Effect of multimer size and a natural dimorphism on the binding of convulxin to platelet glycoprotein (GP)VI.

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.

An association of candidate gene haplotypes and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees.

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.

Analysis of platelet membrane glycoprotein polymorphisms in Glanzmann thrombasthenia showed the French gypsy mutation in the alphaIIb gene to be strongly linked to the HPA-1b polymorphism in beta3.

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.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity.

- 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.

The role of platelet collagen receptor (glycoprotein Ia/IIa; integrin alpha2 beta1) polymorphisms in thrombotic disease.

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.

Characterization of Inherited Differences in Transcription of the Human Integrin alpha 2 Gene.

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.

Allele-dependent transcriptional regulation of the human integrin alpha2 gene.

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.

Platelet receptor polymorphisms and thrombotic risk.

Plaque rupture and/or endothelial damage lead to exposure of von Willebrand factor and collagen which facilitate the adhesion of circulating platelets via glycoprotein Ib-IX-V and the integrin alpha2 beta1, respectively, to the damaged vessel wall. This process activates the platelet and leads to a conformational change of a second integrin alphaIIb beta3 that facilitates fibrinogen binding and platelet aggregation. Thrombin generated at the blood-plaque interface converts fibrinogen to fibrin, which stabilizes thrombus growth. Therefore, any genetic differences that might alter surface expression or activity of these receptors could influence risk for adverse outcomes as a result of the haemostatic process. In the last 5 years, there has been a rapid accumulation of the literature concerning the relationship between genetic variations in platelet glycoproteins and risk for coronary heart disease. In this chapter, we present a comprehensive review of the impact of platelet receptor polymorphisms and thrombotic risk.

Low platelet alpha2beta1 levels in type I von Willebrand disease correlate with impaired platelet function in a high shear stress system.

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.

Association of the platelet glycoprotein Ia C807T gene polymorphism with nonfatal myocardial infarction in younger patients.

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.

A Glanzmann thrombasthenia-like phenotype caused by a defect in inside-out signaling through the integrin alpha(IIb)beta3.

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.

Conformational change in an anti-integrin antibody: structure of OPG2 Fab bound to a beta 3 peptide.

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.

Nucleotide polymorphisms in the alpha2 gene define multiple alleles that are associated with differences in platelet alpha2 beta1 density.

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.

Hereditary variation in platelet integrin alpha 2 beta 1 density is associated with two silent polymorphisms in the alpha 2 gene coding sequence.

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.

Molecular determinants of arg-gly-asp ligand specificity for beta3 integrins.

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.