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Experimental Biology and Medicine 231:505-513 (2006)
© 2006 Society for Experimental Biology and Medicine

MINIREVIEW

Molecular Mechanisms of Prothrombotic Risk Due to Genetic Variations in Platelet Genes: Enhanced Outside-In Signaling Through the Pro33 Variant of Integrin ß3

K. Vinod Vijayan and Paul F. Bray1

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

1 To whom requests for reprints should be addressed at Thrombosis Research Section, Baylor College of Medicine, One Baylor Plaza, BCM 286, N1319, Houston, TX 77030. E-mail: pbray{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Summary
 References
 
In recent years inherited variations in platelet proteins have emerged as potential risk factors that could predispose individuals to arterial thrombosis. Although many studies have examined the association of platelet gene polymorphisms with particular disease states, the underlying mechanisms by which most of these polymorphisms contribute to the pathophysiology of thrombosis have remained largely unexplored. This review will focus on the cellular and molecular features by which these genetic changes affect platelet physiology. Although many genes have been investigated in this regard, only the genes encoding integrins ß3 and {alpha}2, and the platelet Fc receptor, Fc{gamma}RIIA, have been studied in any depth. In some cases (such as integrin {alpha}2), evidence supports a quantitative trait locus. For other genes, nonsynonymous nucleotide substitutions lead to structural and functional consequences. A large portion of this review will focus on the widely studied Leu33Pro (PlA) polymorphism of integrin ß3, and will consider the potential mechanisms by which the Pro33 polymorphism could induce a prothrombotic risk. A detailed understanding of how polymorphisms modulate platelet physiology will be important for understanding individual differences in response to antiplatelet therapy.

Key Words: platelets • genes • polymorphism • integrin • thrombosis • signaling • adhesion • aggregation


   Introduction
Top
 Abstract
 Introduction
Summary
 References
 
Platelets actively participate in vascular thrombosis, resulting in myocardial infarction and stroke, and there is an established genetic component in the pathophysiology of these multifactorial diseases (1–3). Over the past decade much effort has been invested in the study of genetic variations that may modify these disease processes, and several polymorphisms in platelet receptors have been associated with the clinical phenotype. Most experts agree that the genetic risk for the majority of patients with cardiovascular disease amounts to the sum of small effects from numerous different genes (4). Considering the high prevalence and life-threatening potential of cardiovascular disease, the overall significance of even modest effects of prothrombotic platelet polymorphisms may be substantial. Furthermore, the pharmacogenetic interaction between antiplatelet therapies and platelet polymorphisms (5, 6) emphasizes the importance of understanding the effect these inherited variations have on platelet physiology. The clinical aspects of platelet receptor polymorphisms have been reviewed in detail elsewhere (7, 8) and will not be the focus of this review. Rather, we will consider the mechanisms by which polymorphisms may enhance platelet functions, with an emphasis on the nonsynonymous Leu33Pro (PlA) polymorphism of integrin ß3.

Genetic Mechanisms by Which Polymorphisms Affect Platelet Function.
Polymorphisms are stable genetic variations represented in more than 1% of the chromosomes in a given population. Variations in the human genetic sequence occur approximately once every 1,000 base pairs, and there are 200,000 amino acid–altering single nucleotide polymorphisms (SNPs), indicating that genetic variations are a common feature of the human genome (9, 10). Table 1Go summarizes those genes and polymorphisms for which there is evidence for a functional consequence of the genetic alteration. There are two general mechanisms by which DNA polymorphisms might affect platelet function: either by affecting the quantity or the quality of the protein product. The polymorphisms in Table 1Go are grouped according to one of these mechanisms, or if the mechanism is unclear it is listed as "unknown." Only three polymorphisms (one in each of three genes) have been characterized in detail: integrins {alpha}2 and ß3, and Fc{gamma}RIIA. For the remainder, only one or two publications have addressed the physiologic effects of the polymorphism, and the key references are listed in Table 1Go. It should also be appreciated that in some cases it may not be clear whether the polymorphism is itself causative or is in strong linkage disequilibrium (LD) with the causative genetic change.


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  		Table 1. Platelet Gene Polymorphism with Evidence for Altered Platelet Function
 
Quantitative Trail Loci.
Perhaps the simplest means by which a polymorphism would affect platelet function is by altering the quantity of the protein that is expressed by enhancing either transcription or translation. This could occur with polymorphisms in noncoding sequences of a gene. Integrin {alpha}2ß1 (gene ITGA2) is one of the platelet collagen receptors. The laboratory of T.J. Kunicki and coworkers was the first to show that platelets from subjects with the 807T allele have higher levels of {alpha}2ß1, and this has been confirmed by other groups (26–29). Higher levels of {alpha}2ß1 expression correlates with a greater rate of platelet attachment to surfaces coated with collagen Type I under shear stress (27). Thus, carriage of the 807T allele would be predicted to yield a prothrombotic platelet phenotype. The 807T/C SNP is synonymous and likely not responsible for the differences in receptor levels. This SNP is in linkage disequilibrium with several other polymorphisms in the ITGA2 gene (30), including an SNP at –52, which may influence the density of {alpha}2ß1 receptors (12). Thus, it is possible that the 807T polymorphism tracks with the causative SNP at –52, which is responsible for altered ITGA2 transcription and thus, enhanced platelet adhesion to collagen surfaces.

Although the T->C nucleotide substitution at position 1565 in exon 2 of the ß3 gene would not be expected to alter gene expression or protein stability, this has not been formally examined. We have quantified integrin {alpha}2ß1 and {alpha}IIbß3 expression on platelets from 385 healthy human subjects (Fig. 1Go). We confirmed the association between 807T/C and {alpha}2ß1 expression, but observed no significant difference in {alpha}IIbß3 expression according to the Leu33Pro genotype. In addition, we have made a number of different cell lines with each isoform and have no evidence that the Leu33Pro substitution affects protein stability or expression (unpublished data). Thus, it is unlikely that altered {alpha}IIbß3 levels in the Pro33-positive platelets could explain a prothrombotic phenotype for these platelets.


Figure 1
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  		Figure 1. Platelet surface integrin {alpha}2 and ß3 levels by genotype. Levels of integrins {alpha}2ß1 (A) and {alpha}IIbß3 (B) were determined on resting platelets in whole blood from 385 healthy donors using flow cytometry and monoclonal antibodies specific for {alpha}2 and ß3. There were significant differences in {alpha}2 expression between subjects carrying the CC and CT genotype (P < 0.001), CC and TT genotype (P < 0.001), and CT and TT genotype (P < 0.001). The numbers of subjects with CC, CT, and TT genotypes were 141, 169, and 48, respectively. In contrast, no significant differences in ß3 expression between Leu33/Leu33 (L/L) and Leu33/Pro33 (L/P) platelets (P = 0.78), Leu33/Leu33 (L/L) and Pro33/Pro33 (P/P) platelets (P = 0.08), or Leu33/Pro33 (L/P) and Pro33/Pro33 (P/P) platelets (P = 0.11) were noted. There were 305 Leu33/Leu33 subjects, 72 Leu33/Pro33 subjects, and 8 Pro33/Pro33 subjects. Data are mean ± SE of the mean fluorescence intensity.

 
Amino Acid Substitutions Altering Function.
If polymorphisms occur in the coding sequence of the gene and alter an amino acid, a change in the structure, antigenicity, or function of the encoded protein (or a combination of these) is possible.

Fc{gamma}RIIA His131Arg.
Platelets have a single Fc receptor, Fc{gamma}RIIA. This receptor is also expressed on T lymphocytes, where it has variable ability to induce mitogenesis after binding mouse IgG1 (31), depending upon whether residue 131 is a histidine or an arginine (32). Compared with the Arg131 isoform, the His131 form binds murine IgG1 with lower affinity than does the Arg131 form; the Arg131 form binds human IgG2 poorly (33). We have shown that this polymorphism of FCGR2A does not affect intrinsic platelet reactivity, but that some activation-dependent platelet monoclonal antibodies bind preferentially to Arg131-positive platelets, cross-link Fc{gamma}RIIA, and activate platelets (34). Thus, assays of cellular responsivity using monoclonal antibodies and cells expressing Fc{gamma}RIIA must take caution to use Fab fragments, block the Fc{gamma}RIIA, or otherwise consider the His131Arg genotype in the data analysis (34).

Integrin ß3 Leu33Pro (PlA).
The Leu33Pro polymorphism of integrin (glycoprotein IIIa) was originally identified because of its prominence in causing alloimmune thrombocytopenias (35). Newman et al. (36) identified the genetic basis for this polymorphism, which induces a Leu33Pro substitution, and which alters integrin ß3 antigenicity. This study also demonstrated that Leu33 was responsible for the PlA1 epitope, and Pro33 for the PlA2 epitope. The prevalence of the PlA1 and PlA2 alleles varies by ethnic background and geographical distribution. The common allele is PlA1 with reported frequencies of 0.84–0.89 in whites, 0.92 in blacks, and > 0.99 in Asians. The PlA2 allele is not rare in certain populations, occurring with a frequency of 0.11–0.15 in whites and 0.08 in blacks. However, the PlA2 allele is nearly absent in Asians (< 0.01). This polymorphism was also the first platelet variation linked to myocardial infarction (37), findings that have led to a plethora of subsequent genetic epidemiology studies in this area. Meta-analysis studies concluded that the Pro33 polymorphism offers a mild risk for myocardial infarction but not stroke, especially for younger subjects and those undergoing revascularization procedures (38, 39). As will be explained, the weight of the evidence suggests this polymorphism affects integrin ß3 function.

Linkage Disequilibrium with a Causative Gene.
Linkage disequilibrium refers to the phenomenon wherein a combination of alleles occurs more frequently than what would be predicted by a chance. LD between alleles occurs because of infrequent chromosomal crossover between genes during meiosis. For any polymorphism that has been associated with a clinical or physiologic phenotype, it should always be considered that it is merely in LD with (i.e., a "marker" for) the causative variation, such as those described above. For example, it is conceivable that the mere presence of C at 1565 on ß3 is not directly responsible for the observed prothrombotic phenotype in the Pro33-positive platelets, but rather the ß3 gene containing 1565 C may be in LD with a different causative variation in the same or different gene. To date, there is no evidence to support the possibility of this polymorphism.

Physiologic Consequences of the Leu33Pro Polymorphism of Integrin ß3.
There is strong immunologic evidence that the Leu33Pro substitution alters protein structure, because each antigen can induce an immune response in individuals lacking that antigen. The initial evidence suggesting that this structural change may affect platelet function came from observations that most anti-Leu33 antibodies block fibrinogen binding and inhibit aggregation of Leu33/Leu33 platelets, and retard or partially inhibit aggregation of Pro33/Leu33 platelets (40), despite the linear distance between position 33 and the putative fibrinogen binding sites (amino acids 109–171 and 211–222) on integrin ß3 (41, 42). This issue has been examined formally in subsequent studies with platelets and stably transfected cell lines.

Studies with Platelets.
In vivo studies of platelet function found Pro33-positive subjects have significantly shorter bleeding times, and enhanced thrombin generation and prothrombin consumption than Pro33-negative subjects (5, 43, 44). The largest study of ex vivo platelet function was the Framingham Offspring Study, which included 1422 subjects (45), and which considered confounders in their analyses. This study found that significantly less epinephrine was required to induce platelet aggregation in the Pro33-positive than in Pro33-negative platelets, and this effect was PlA2 allele dose dependent (i.e., a dominant effect) (Table 2Go). A similar trend was observed when platelets were stimulated with ADP. We have found that compared with Pro33-negative platelets, Pro33-positive platelets exhibit increased {alpha} granule secretion in response to ADP and thrombin (5, 46). Other studies using small numbers of subjects have obtained inconsistent results, perhaps due to differences in the assays, study design, or variation in donor characteristics that might affect platelet function (6, 47–51). Considering the marked variation in platelet function between individuals, it is difficult to interpret the results of small studies that do not consider confounding variables such as age, gender, etc. But taken together, the bulk of the available evidence suggests that the Pro33 polymorphism of integrin ß3 can alter human platelet aggregation, adhesion, and secretion.


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  		Table 2. Association of the Leu33Pro Polymorphism with Platelet Aggregationa
 
Studies with Heterologous Cells.
To overcome the limitation caused by heterogeneity of human platelets, model systems including Chinese Hamster Ovary (CHO) and human kidney embryonal 293 cell lines overexpressing either the Leu33 or Pro33 isoforms of {alpha}IIbß3 have been utilized (52). These cells were used to study integrin outside-in signaling functions such as adhesion, spreading, migration, and clot retraction. Compared with the Leu33 cells, a significant 30%–60% increase in {alpha}IIbß3-mediated adhesion and haptotactic migration of the Pro33 cells to immobilized fibrinogen under static conditions was observed (52, 53). Pro33 cells also displayed increased fibrin clot retraction. We performed these assays only when the Leu33 and Pro33 cell lines had equivalent levels of {alpha}IIbß3 expression. Another group found no effect of the polymorphism on static adhesion to fibrinogen using a lymphocyte cell line, GM1500, following stimulation with exogenous phorbol myristate acetate (51). Figure 2Go shows how compared with Leu33 cells, fluid shear stress augmented the difference in adhesiveness of Pro33 cells to fibrinogen by 1.7-fold to 4.5-fold (54). Adhesion under shear was integrin {alpha}IIbß3-mediated and substrate specific, because the increased Pro33 adhesiveness was observed with von Willebrand factor (VWF) and cryoprecipitate (composed of VWF and fibrinogen) but not fibronectin (54). Confocal microscopy revealed a greater spreading and extensive actin polymerization of the Pro33 cells compared with the Leu33 cells on fibrinogen substrate (Fig. 3Go) (52). In addition, compared with the Leu33 cells, Pro33-expressing {alpha}vß3 cells had an enhanced haptotactic migratory response to vitronectin and osteopontin but not to fibrinogen, suggesting that the {alpha} subunit influences the substrate specificity of this genetic effect (53). In summary, studies with human platelets and heterologous cells demonstrate that the {alpha}IIbß3-dependent functions of aggregation, secretion, adhesion, spreading, migration, and clot retraction are increased by the Pro33 isoform (Fig. 4Go).


Figure 2
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  		Figure 2. Adhesion of Leu33 and Pro33 CHO cells on fibrinogen under fluid shear stress. (A) Cells were perfused over 100 µg/ml fibrinogen in a parallel plate flow chamber and the number of adherent cells quantified. LK is the control CHO cells transfected with LK444 parental vector. Compared with Leu33 cells, Pro33 cells show 4.5-fold increased adhesion at 25 secs–1, 1.7-fold increased adhesion at 50 secs–1, and 2.6-fold increased adhesion at 100 secs–1. The increased adhesion of Pro33 over Leu33 was significant at *P < 0.001. (B) Mean fluorescence intensity of integrin {alpha}IIbß3 was detected using P2 monoclonal antibody to the three cell lines; n = 3. Reproduced with permission following modification (54).

 

Figure 3
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  		Figure 3. Confocal microscopy (A) following rhodamine phallodin staining of CHO cells that adhered to a fibrinogen-coated coverslip for 5 mins. Compared with the Leu33 cells an increased F-actin staining was observed for the Pro33 cells. (B) Following 15 mins of incubation on fibrinogen, greater spreading of the Pro33 cells was observed. The morphometric analysis of 35–50 cells revealed a significant increase (P < 0.001 and P = 0.01) for F actin and spreading by the Pro33 cells, respectively. Reproduced with permission following modification (52).

 

Figure 4
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  		Figure 4. Functional consequences of the Pro33 isoform of integrin ß3. Fibrinogen binding by the Pro33 isoform of integrin ß3 results in an increased {alpha}IIbß3-mediated function in platelets and heterologous cells.

 
Potential Cellular and Molecular Mechanisms for the Prothrombotic Phenotype Associated with the Pro33 Polymorphism.
How could the structural alteration of the Pro33 isoform confer increased {alpha}IIbß3 function? In this section we will consider an approach to evaluating the potential mechanisms by which any non-synonymous polymorphism might induce a prothrombotic platelet phenotype, using the Pro33 isoform of integrin ß3 as a prototype. A few factors that are likely to play a critical role are discussed below.

Increased Affinity for Fibrinogen by the Pro33 Isoform of {alpha}IIbß3.
It is conceivable that the conformational change induced by the Pro33 isoform of integrin {alpha}IIbß3 could increase the affinity of the receptor for its ligands. However, Bennett et al. (51) observed no significant difference in the affinity of fibrinogen binding or in maximal fibrinogen binding between the Pro33-positive and the Pro33-negative platelets. The concentration of fibrinogen producing half-maximal binding was not significantly different for the Pro33-positive and the Pro33-negative CHO cells, suggesting that the binding kinetic of soluble fibrinogen was not altered by the Pro33 isoform (52). Thus, these two studies found no evidence that the Pro33 isoform alters the affinity of the {alpha}IIbß3 for fibrinogen. It is somewhat difficult to reconcile this conclusion with the enhanced agonist-induced platelet aggregation observed in the Framingham study (45). Furthermore, when we purified {alpha}IIbß3 from human platelets of known Leu33Pro genotype we found that purified human Pro33-positive integrins exhibited an enhanced binding to immobilized fibrinogen and prothrombin,1 suggesting an intrinsic difference in the Pro33 isoform that favors fibrinogen binding. Thus, the possibility that the Pro33 isoform alters affinity or otherwise enhances inside-out signaling to {alpha}IIbß3 remains unclear.

Increased {alpha}IIbß3 Avidity by the Pro33 Isoform.
Li et al. (55) have shown that integrins cluster after activation, and the loop of integrin ß3 containing residue 33 is extended to a position that would allow it to participate in receptor oligomerization (56). Thus, it is possible that the Pro33 isoform could alter the avidity of the {alpha}IIbß3 via increased clustering. To date, no studies have examined whether the Leu->Pro 33 change affects the avidity of {alpha}IIbß3. However, the distribution patterns of integrin {alpha}IIbß3 as detected by fluorescence microscopy were similar in Leu33 and Pro33 CHO cells that adhered to fibrinogen (54). This observation may indirectly suggest that the enhanced adhesion of the Pro33 cells to fibrinogen is not caused by extensive integrin clustering.

Differential Association of Transmembrane Signaling Molecules by the Pro33 Isoform of {alpha}IIbß3.
Integrin {alpha}IIbß3 has been shown to associate with transmembrane molecules, including some that transduce signals, such as Fc{gamma}RIIa, which contains an immunoreceptor tyrosine-based activation motif; platelet-derived growth factor receptor ß; and integrin-associated protein and tetraspannin family members CD9 and CD63 (57–60). The conformational change in the extracellular region of integrin ß3 in the Pro33-positive platelets could alter its interaction with some of these transmembrane signaling molecules. None of the published studies on the Pro33 polymorphism have addressed this possibility. In unpublished studies we could not detect an association between {alpha}IIbß3 and platelet endothelial cell adhesion molecule-1 (which contains an immunoreceptor tyrosine-based inhibitor motif) that was dependent upon the Leu33Pro genotype. Additional studies are needed to clarify whether the Pro33 isoform modulates the interaction of transmembrane signaling and adhesion molecules.

Increased {alpha}IIbß3 Outside-In Signaling by the Pro33 Isoform.
Ligand binding to {alpha}IIbß3 induces outside-in signaling, which results in rearrangement of the platelet actin cytoskeleton and activation of signaling molecules (61). Rearrangement of the platelet cytoskeleton is essential for platelet shape change, spatial regulation of receptors, creation of membrane microdomains, and recruitment of several kinases and phosphatases. Integrin outside-in signaling results in the phosphorylation of multiple proteins including the focal adhesion kinase (FAK), extracellular-signal regulated kinase 2 (ERK2), and myosin light chain (MLC). These functions collectively integrate into increased platelet reactivity.

Signaling to FAK, ERK2, and MLC.
Compared with Leu33 cells, adhesion of Pro33 CHO or 293 cells (or both cell types) to immobilized fibrinogen shows increased phosphorylation of FAK at Tyr125, ERK2 at Thr183 and Tyr185, and MLC at Thr18 and Ser19 (52, 62). The increased adhesiveness and migration of the Pro33 CHO cells is abolished by inhibiting actin cytoskeleton rearrangement with cytochalasin-D (52–54). The {alpha}IIbß3 outside-in signaling changes exhibited by the Pro33 isoform are also observed with human platelets after adhesion to fibrinogen and thrombin-induced aggregation (46, 62). The enhanced cellular function (adhesion, migration, and clot retraction) exhibited by the Pro33 CHO cells are abolished by inhibiting ERK2 phosphorylation with mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126 (46, 53). These results are consistent with the established role of ERK2 signaling in regulating cell adhesion, spreading, and haptotactic and chemotactic cell migration, (63–66).

MLC phosphorylation promotes myosin ATPase activity and increases the actinomyosin contractile response that is involved in platelet shape change, secretion, ADP-induced aggregation, and migration (67, 68). Pro33-positive platelets exhibit increased platelet MLC phosphorylation and enhanced {alpha} granule (P-selectin) secretion. Blocking MLC phosphorylation abolishes the increased adhesiveness of the Pro33 cells and increased secretion of the Pro33-positive platelets, suggesting that signaling through MLC mediates the enhanced activity of Pro33-expressing cells and platelets (46, 62).

Role for Serine-Threonine Phosphatases in Increased MLC Phosphorylation in Pro33-Positive Platelets.
The enhanced outside-in signaling in Pro33-positive platelets is independent of integrin ß3 phosphor-ylation at Tyr747, Tyr759, or adaptor protein Shc phosphorylation at Tyr317 (62). These phosphorylation events are believed to be critical in {alpha}IIbß3 outside-in signaling and ERK2 phosphorylation (69, 70). The phosphorylation state of platelet signaling molecules at any time is a composite of agonist-mediated phosphorylation and outside-in integrin-mediated dephosphorylation. Figure 5Go shows that when platelets are stimulated with thrombin, MLC shows much greater phosphorylation in the Pro33-positive platelets than in the Pro33-negative platelets (62). However, when the {alpha}IIbß3-fibrinogen interaction is blocked, MLC phosphorylation dramatically increases in the Pro33-negative platelets, with a minor effect on Pro33-positive platelets, suggesting that integrin engagement negatively regulates MLC phosphorylation, and this process is modified by the Pro33 isoform. The Ser/Thr phosphatase inhibitor okadaic acid enhances ERK2 and MLC phosphorylation more efficiently in the Pro33-negative platelets, indicating that a phosphatase is involved in the Pro33-dependent effects on MLC and ERK2. Taken together, these signaling studies indicate: (i) {alpha}IIbß3 engagement activates Ser/Thr phosphatases, and (ii) residue 33 of integrin ß3 regulates the activation of Ser/Thr phosphatases. Furthermore, Pro33-positive platelets exhibit an increased phosphorylation of the PP1-myosin phosphatase (MP) that dephosphorylates MLC (62). This phosphorylation occurs at Thr696 in the myosin binding subunit of MP, thus blocking the MP activity. Thus, compared with the Leu33-positive platelet MP activity is inactivated in aggregating Pro33-positive platelets. The model shown in Figure 6Go indicates how the increased MLC phosphorylation observed during the aggregation of Pro33-positive platelet is due in part to inefficient activation of a serine/threonine phosphatase via increased Thr696 phosphorylation.


Figure 5
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  		Figure 5. Increased MLC phosphorylation in Pro33-positive platelets. Leu33-positive or Pro33-positive platelets were aggregated using varying concentrations of thrombin and blotted with antiphospho MLC (ppMLC) or total MLC. Reproduced with permission following modification (62).

 

Figure 6
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  		Figure 6. Model for increased MLC phosphorylation in Pro33-positive platelets. Platelet agonists such as thrombin induce robust phosphorylation of MLC. In contrast, {alpha}IIbß3-fibrinogen interaction leads to dephosphorylation of MLC. Thus, the phosphorylation pattern of MLC during thrombin-induced platelet aggregation is a composite of both these signaling events. Blocking {alpha}IIbß3-fibrinogen interaction or inhibiting myosin phosphatase (MP) blocks the dephosphorylation of MLC during aggregation, suggesting that {alpha}IIbß3-fibrinogen interaction activates a serine/threonine phosphatase. However, in case of the Pro33-positive platelets, {alpha}IIbß3-fibrinogen interaction also results in an increased phosphorylation of MP at Thr696. Phosphorylated MP is inactive, and therefore, compared with the Pro33-negative platelets, integrin engagement in the Pro33-positive platelets does not effectively dephosphorylate MLC, resulting in an increased MLC phosphorylation. Reproduced with permission of the publisher following modification (62).

 
Clues from {alpha}IIbß3 Crystal Structure.
The extra-cellular domains of recombinant {alpha}IIbß3 have now been crystallized. Can we glean any clues from this structure information that might help us understand the functional and signaling effects regulated by the leucine or proline at residue 33 of integrin ß3? Xiao et al. (56) have identified three conformations of extracellular {alpha}IIbß3: (i) a low-affinity, bent conformation; (ii) an extended form of intermediate affinity with the two N-terminal headpieces in a closed conformation; and (iii) a ligand-bound conformation with {alpha}IIb and ß3 extended, but with the headpieces open and in a high-affinity state. The headpiece of ß3 consists of the ßI, hybrid, and plexin-semaphorin-integrin (PSI) domains. PSI domains are conserved in other species and known to bind other proteins, and there is evidence to indicate that the PSI domain participates in integrin activation (71). The Leu33 residue of ß3 is in the long and flexible loop of the PSI domain. Xiao et al. proposed that after ligand binding the rigid nature of the hybrid and PSI domains causes a widening of the angle between the {alpha} and ß legs that favors leg extension and separation. The substitution of a helix-favoring residue Leu with a helix-disrupting residue Pro is likely to alter the local conformation of integrin ß3—particularly because there is a Pro at position 36. Assuming the Pro33 isoform of ß3 causes no major structural alterations, a PSI domain containing a Pro33 might be more rigid, which could favor leg separation, or provide greater stability to the open-head conformation after ligand binding than would the Leu33 isoform (or both). Such changes might favor outside-in signaling.

It is also noteworthy that the Leu33 residue is contained within three disulfide loops: Cys13-Cys435, Cys16-Cys38, and Cys26-Cys49 (56). Because disulfide bonds form in the endoplasmic reticulum during synthesis of the nascent peptide, it is not certain that a Pro33 isoform would fold and assemble disulfides such as Leu33. So despite invaluable information derived from the crystal structure of Leu33 ß3, until a Pro33 structure is solved, we can only speculate about its structure and how this might affect function.


   Summary
Top
 Abstract
 Introduction
 Summary
References
 
There is abundant evidence that inherited variations in platelet genes affect platelet physiology by quantitative or qualitative effects on important platelet adhesive and signaling molecules. It may be expected that individuals who inherit multiple prothrombotic genetic effects—even if each individual variant has a relatively small effect on platelet physiology—will be at increased risk for clinical thrombotic events. The extensively studied Pro33 polymorphism of integrin ß3 has been associated with adverse cardiovascular events in many studies. Extensive experimentation using cell lines and platelets demonstrates that the Pro33 polymorphism imparts a prothrombotic platelet phenotype, with (i) enhanced platelet aggregation and secretion; (ii) increased cell adhesion, spreading, migration, and clot retraction; and (iii) greater {alpha}IIbß3 outside-in signaling. On the basis of available data it appears that compared with the Leu33 isoform, postreceptor occupancy with the Pro33 isoform results in a rapid remodeling into a highly ordered cytoskeletal structure that supports the recruitment and activation of signaling molecules such as FAK, ERK2, and MLC, thereby modulating integrin function. Investigation into the molecular mechanisms has revealed that inefficient activation of Ser/Thr phosphatases is responsible in part for the efficient {alpha}IIbß3 outside-in signaling in the Pro33-positive platelets. Thus, although the effect of the Pro33 polymorphism on inside-out signaling and {alpha}IIbß3 affinity is still unclear, an emerging concept is that this common genetic variation contributes to increased signaling and cytoskeletal changes, thereby reinforcing platelet responses to postactivation events, and stabilizing platelet-platelet or platelet-extracellular matrix interactions.


   Footnotes
 
K.V.V. is supported by National Scientist Development Grant 0435017N from the American Heart Association. P.F.B. is supported by grants HL65229 and HL65967 from the National Institutes of Health.

1 Vijayan KV, Bray PF. Unpublished data. Back


   References
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 Introduction
 Summary
 References
 

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