Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/FcRgamma-chain complex from the human platelet surface

Platelet glycoprotein (GP) VI is a 62-kDa membrane glycoprotein that exists on both human and murine platelets in a noncovalent complex with the Fc receptor (FcR) gamma chain. The GPVI/FcRgamma-chain complex serves as the major activating receptor for collagen, as evidenced by observations that platelets genetically deficient in GPVI or the FcRgamma chain are highly refractory to collagen-induced platelet activation. Recently, several different rat anti-murine GPVI monoclonal antibodies, termed JAQs 1, 2, and 3, were produced that had the unique property of "immunodepleting" GPVI from the murine platelet surface and rendering it unresponsive to collagen or GPVI-specific agonists like convulxin or collagen-related peptide (CRP). Herein, we describe a patient with a mild bleeding disorder and a moderately reduced platelet count whose platelets fail to become activated in response to collagen or CRP and inefficiently adhere to and form thrombi on immobilized collagen under conditions of arterial shear. Although the amount of GPVI platelet mRNA and the nucleotide sequence of the GPVI gene were found to be normal, both GPVI and the FcRgamma chain were nearly absent from the platelet surface and were markedly reduced in wholeplatelet detergent lysates. Patient plasma contained an autoantibody that bound specifically to GPVI-positive, normal platelets, and cleared soluble GPVI from the plasma, suggesting that the patient suffers from a rare form of idiopathic thrombocytopenic purpura caused by a GPVI-specific autoantibody that mediates clearance of the GPVI/FcRgamma-chain complex from the platelet surface. Since antibody-induced GPVI shedding now has been demonstrated in both humans and mice, these studies may provide a rationale for developing therapeutic reagents that induce temporary depletion of GPVI for the treatment of clinical thrombosis.     

The influence of N-linked glycosylation on the function of platelet glycoprotein VI

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.     

Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase

Thrombosis can be initiated when activated platelets adhere to injured blood vessels via the interaction of subendothelial collagen with its platelet receptor, glycoprotein (GP) VI. Here we observed that incubation of platelets with convulxin, collagen, or collagen-related peptide (CRP) resulted in GPVI signaling-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble fragment of GPVI as revealed by immunoblotting. Ethylenediaminetetraacetic acid (EDTA) or GM6001 (a metalloproteinase inhibitor with broad specificity) prevented this loss. In other receptor systems, calmodulin binding to membrane-proximal cytoplasmic sequences regulates metalloproteinase-mediated ectodomain shedding. In this regard, we have previously shown that calmodulin binds to a positively charged, membrane-proximal sequence within the cytoplasmic tail of GPVI. Incubation of platelets with calmodulin inhibitor W7 (150 microM) resulted in a time-dependent loss of GPVI from the platelet surface. Both EDTA and GM6001 prevented this loss. Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI. These findings suggest that disruption of calmodulin binding to receptor cytoplasmic tails by agonist binding to the receptor triggers metalloproteinase-mediated loss of GPVI from the platelet surface. This process may represent a potential mechanism to regulate GPVI-dependent platelet adhesion.     

Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood

The platelet glycoproteins (GPs) Ib, integrin alpha(2)beta(1), and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s(-1)), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb alpha (12G1 Fab(2)) and alpha(2)beta(1) (6F1 mAb or GFOGER-GPP peptide). GPVI was found to be crucial for aggregate formation, Ca(2+) signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting alpha(2)beta(1) revealed its involvement in regulating Ca(2+) signaling, PS exposure, and aggregate size. Both GPIb alpha and alpha(2)beta(1) contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb alpha or alpha(2)beta(1). In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with alpha(2)beta(1) mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.     

Collagen mediates changes in intracellular calcium in primary mouse megakaryocytes through syk-dependent and -independent pathways.

We have characterized changes in [Ca2+]i in primary mouse megakaryocytes in response to fibrillar collagen and in response to cross-linking of the collagen receptor, the integrin alpha2beta1. The response to collagen was markedly different from that seen to a triple helical collagen-related peptide (CRP), which signals via the tyrosine kinases p59(fyn) and p72(syk). This peptide binds to the collagen receptor glycoprotein VI (GPVI), but not to the integrin alpha2beta1. Collagen elicited a sustained increase in [Ca2+]i composed primarily of influx of extracellular Ca2+ with some Ca2+ release from internal stores. In contrast to CRP, this response was only partially (approximately 30%) inhibited by the src-family kinase inhibitor PP1 (10 micromol/L) or by microinjection of the tandem SH2 domains of p72(syk). Collagen also caused an increase in [Ca2+]i in megakaryocytes deficient in either p59(fyn) or p72(syk), although the response was reduced by approximately 40% in both cases: Cross-linking of the alpha2 integrin increased [Ca2+]i in these cells exclusively via Ca2+ influx. This response was reduced by approximately 50% after PP1 pretreatment, but was significantly increased in fyn-deficient megakaryocytes. Collagen therefore increases [Ca2+]i in mouse megakaryocytes via multiple receptors, including GPVI, which causes Ca2+ mobilization, and alpha2beta1, which stimulates a substantial influx of extracellular Ca2+.     

Molecular priming of Lyn by GPVI enables an immune receptor to adopt a hemostatic role

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates.The response of platelets to vessel injury is essential to prevent posttraumatic blood loss but also underlies the pathophysiology of cardiovascular diseases, such as heart attack and stroke (1). Platelet activation requires the generation of rapid and coordinated intracellular signals that culminate in cell-matrix and cell-cell adhesion and thrombus formation within seconds (2). Collagen, the most abundant and thrombogenic subendothelial matrix protein exposed by vessel injury, provides both a primary activating stimulus and an adhesive surface for the initiation of platelet thrombi in the arterial system. Understanding these events has therapeutic potential, as blocking this early step may prevent the formation of pathologic thrombi associated with the coronary and cerebral arteries (3).Molecular and genetic studies have established that collagen-induced platelet activation is triggered by the platelet-specific surface receptor glycoprotein VI (GPVI). GPVI is an Ig (Ig)-like domain-containing receptor that is structurally and functionally homologous to immune receptors but is expressed exclusively on platelets and megakaryocytes (4). Extensive pharmacologic and genetic studies have demonstrated that GPVI signaling in response to collagen is highly analogous to that of the related multisubunit T-cell receptor and the IgE and IgA receptors (5). GPVI ligands, such as collagen or the snake-venom toxin convulxin, induce receptor clustering that facilitates the phosphorylation of the tandem tyrosines found in the immunotyrosine activating motif (ITAM) of the noncovalently associated FcRγ chain adaptor by Src-family tyrosine kinases (SFKs), such as Lyn and Fyn. ITAM phosphorylation stimulates the recruitment and activation of the intracellular tyrosine kinase Syk and the assembly of a signaling complex that includes the adaptors linker for activated T-cells (LAT) and Src homology (SH)2 domain-containing leukocyte phosphoprotein of 76 kDa (Slp-76), culminating in the activation of phospholipase C (PLC)γ2 (5, 6). This pathway, like the established G protein-coupled signaling pathways that mediate platelet activation by thrombin and ADP, results in the elevation of intracellular calcium levels and platelet activation responses, including granule release and integrin conformational changes. In contrast to immune receptors that form stable immune synapses and transmit signals, which generate cellular responses over several hours, platelet activation by GPVI takes place within seconds, allowing circulating platelets to firmly adhere to exposed collagen (7, 8). Whether and how platelets have adapted the immune receptor homologue GPVI to meet the accelerated temporal requirements inherent in hemostasis is unknown.GPVI, like homologous immune receptors, contains conserved transmembrane residues that are required for its association with the FcRγ chain, the essential signal transducing component of the receptor complex (9). However, a feature of GPVI that is unique among FcRγ-coupled receptors is a conserved intracellular class I (R/K)xxPxxP proline-rich domain (PRD) of the amino acid sequence K/RPLPPLP that constitutes a core binding motif for SH3 domains, such as those found on SFKs (10) [supporting information (SI) Fig. S1A]. Biochemical studies in cell lines have demonstrated that the GPVI PRD mediates binding to the SFKs Lyn and Fyn (11, 12). These studies have demonstrated variable degrees of reduced signaling by GPVI receptors lacking this motif (11, 13). However, the role of the GPVI PRD in platelets and hemostasis has not been defined.To study the function of the GPVI PRD, we generated mice with platelets that expressed wild-type GPVI or GPVI lacking the PRD. Our studies reveal that the PRD does not alter the strength of sustained GPVI signaling but instead accelerates platelet activation kinetics, a feature required for the efficient adhesion of platelets to collagen under flow conditions. Molecular studies to explain the basis for signal acceleration by the GPVI PRD reveal that this domain specifically binds the SH3 domain of Lyn. We find that GPVI PRD binding directly activates Lyn and that GPVI-bound Lyn in resting platelets is in an active state before receptor ligation. These studies provide insight into the molecular mechanism by which platelets have adapted the immune-receptor signaling pathway to function under the temporal constraints of the hemostatic system.     

Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin alpha(2)beta(1) was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc(2)) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc(2) bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils.     

GPVI levels in platelets: relationship to platelet function at high shear

We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and alpha 2 beta 1 on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of alpha 2 beta 1, which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and alpha 2 beta 1 levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor gamma-chain (FcR gamma-chain) complex or a functional FcR gamma-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s-1 and 1500 s-1. These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear.     

Intracellular localization of glycoprotein VI in human platelets and its surface expression upon activation

Glycoprotein (GP) VI is a major receptor for collagen and belongs to the immunoglobulin super family. Here, we examined the localization of GPVI in resting and activated human platelets by immunogold scanning and transmission electron microscopy and flow cytometry. Ultrastructural observation detected immunolabelling for GPVI that was distributed uniformly over the entire surface of resting platelets, and revealed that GPVI was also localized on both the membranes of the surface-connected open canalicular system (OCS) and alpha-granules. The OCS- and alpha-granule-associated GPVI pools were an estimated 35.4 +/- 3.2% (mean +/- standard deviation) of the total. Little GPVI labelling was observed in any part of GPVI-deficient platelets. A remarkable time-dependent increase in GPVI surface expression was found by flow cytometry when platelets were activated by collagen-related peptide (CRP) and convulxin. The GPVI-mediated activation of platelets by CRP or convulxin resulted in similar ultrastructural changes and an increased GPVI labelling density on the activated platelet surface, which was accompanied by a decreased interior expression. GPVI was also expressed on microparticles generated from activated platelets. Thus, our study demonstrates that platelets have internal pools of GPVI, and that GPVI is increasingly redistributed to the surface membrane and to microparticles during platelet activation.     

Murine GPVI stimulates weak integrin activation in PLCgamma2-/- platelets: involvement of PLCgamma1 and PI3-kinase

Collagen stimulates platelet activation through a tyrosine kinase-based pathway downstream of the glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex. Genetic ablation of FcR gamma-chain results in a complete inhibition of aggregation to collagen. In contrast, a steady increase in light transmission is induced by collagen in phospholipase Cgamma2-deficient (PLCgamma2-/-) platelets in a Born aggregometer, indicating a weak level of activation. This increase is inhibited partially in the presence of an alpha2beta1-blocking antibody or an alphaIIbbeta3 antagonist and completely by a combination of the 2 inhibitors. It is also abolished by the Src kinase inhibitor PP1 and reduced in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. The GPVI-specific agonists convulxin and collagen-related peptide (CRP) also stimulate weak aggregation in PLCgamma2-/- platelets, which is inhibited by wortmannin and PP1. Collagen and CRP stimulate tyrosine phosphorylation of PLCgamma1 at its regulatory site, Tyr 783, in murine but not in human platelets through a Src kinase-dependent pathway. Adhesion of PLCgamma2-/- platelets to a collagen monolayer is severely reduced at a shear rate of 800 s-1, relative to controls, whereas it is abolished in FcR gamma-chain-/- platelets. These results provide strong evidence that engagement of GPVI stimulates limited integrin activation in PLCgamma2-/- platelets via PLCgamma1 and PI3-kinase.     

Tec regulates platelet activation by GPVI in the absence of Btk

The Tec family kinase Btk plays an important role in the regulation of phospholipase C gamma 2 (PLC gamma 2) downstream of the collagen receptor glycoprotein VI (GPVI) in human platelets. Platelets also express a second member of this family, Tec; however, its function has not been analyzed. To address the role of Tec, we analyzed Btk-/-, Tec-/-, and Btk/Tec double-deficient (Btk-/-/Tec-/-) platelets. Tec-/- platelets exhibit a minor reduction in aggregation to threshold concentrations of collagen or the GPVI-specific agonist collagen-related peptide (CRP), whereas responses to higher concentrations are normal. Tyrosine phosphorylation of PLC gamma 2 by collagen and CRP is not altered in Tec-/- platelets. However, Btk-/-/Tec-/- platelets exhibit a greater reduction in PLC gamma 2 phosphorylation than is seen in the absence of Btk, thus revealing an important role for Tec in this situation. Furthermore, Btk-/-/Tec-/- platelets fail to undergo an increase in Ca2+, aggregation, secretion, and spreading in response to collagen or CRP, whereas they aggregate normally to adenosine diphosphate (ADP) and spread on fibrinogen. A residual GPVI signal exists in the Btk-/-/Tec-/- platelets as CRP synergizes with ADP to mediate aggregation. These results demonstrate an essential requirement for Tec and Btk in platelet activation by GPVI and reveal a functional role for Tec in the regulation of PLC gamma 2 in the absence of Btk.     

Platelet GPVI binds to collagenous structures in the core region of human atheromatous plaque and is critical for atheroprogression in vivo

Platelet adhesion to the atherosclerotic vascular wall induces thrombosis and boosters vascular inflammation and atheroprogression. In the present study we studied the binding of the platelet collagen receptor glycoprotein (GP) VI to human atherosclerotic plaques (AP) and the role of GPVI-mediated platelet adhesion for atheroprogression. Soluble GPVI-Fc fusion protein bound to immobilized collagen type I, collagen type III, and predominantly to the core region of human carotid atheromatous plaques. The pattern of GPVI-Fc binding was similar to the immunostaining pattern of collagen type III and differed from the immunostaining of collagen type I, which was more intense in the cap than in the core. Plaque-induced platelet aggregation in stirred blood and platelet adhesion/aggregate formation under flow were inhibited by the anti-GPVI monoclonal antibody 5C4 or by pretreatment of plaques with anti-collagen type I and anti-collagen type III antibody, or GPVI-Fc. However, there was no correlation between GPVI-Fc binding and platelet aggregating activity of individual plaques. GPVI bound also to atherosclerotic arteries of ApoE-deficient mice in vivo as assessed by small animal positron emission tomography (PET). Prolonged administration of soluble GPVI attenuated atheroprogression in ApoE-deficient mice. In humans, GPVI binding to collagenous type I and type III structures of the plaque core region mediates plaque-induced platelet adhesion and aggregation, but GPVI binding is not the sole platelet-activating determinant of plaques. In mice, GPVI-mediated platelet adhesion to the atherosclerotic vascular wall is involved in atheroprogression in vivo. Taken together, our data suggests that GPVI is a relevant target to prevent atherothrombotic events and atheroprogression.     

The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression

Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P <.0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P <.001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer-100 (t test, P =.065 and P =.061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.     

The role of selenocysteine 133 in catalysis by the human type 2 iodothyronine deiodinase

Human type 2 iodothyronine deiodinase (hD2) catalyzes the activation of T4 to T3. D2, like types 1 and 3 deiodinases, contains selenocysteine (Sec) in the highly conserved active center at position 133. To evaluate the contribution of Sec133 to the catalytic properties of hD2, we generated mutants in which cysteine (Cys) or alanine (Ala) replaced Sec133. The Km (T4) of Cys133D2 was 2.1 microM, strikingly higher than that of native D2 (1.4 nM). In contrast, the relative turnover number was 10-fold lower for Cys133D2, illustrating the greater potency of Se than S in supporting catalysis. The AlaD2 mutant was inactive. Studies in intact cells transiently expressing the native or Cys133D2 enzyme exhibited saturation kinetics expected from the Km as measured under in vitro conditions, indicating rapid equilibration of extracellular and intracellular T4. Blockade of the NTCP, OATP1-3, and LST-1 transporters with 10 mM sodium taurocholate did not alter the deiodination rate of T4 by Cys133D2 transiently expressed in intact cells, suggesting that intracellular transport of T4 is not rate limiting. These results illustrate that selenium plays a critical role in deiodination catalyzed by hD2.     

The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes.     

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin,but neither is required for regulation of phospholipase C

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.     

Structural basis for platelet collagen responses by the immune-type receptor glycoprotein VI

Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immune-type receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.     

Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

OBJECTIVE: Previous studies have shown the importance of somatic mutations and arginine residues in the complementarity-determining regions (CDRs) of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies in human and murine lupus, and in studies of murine antibodies, a role of mutations at position 53 in V(H) CDR2 has been demonstrated. We previously demonstrated in vitro expression and mutagenesis of the human IgG1 monoclonal antibody B3. The present study was undertaken to investigate, using this expression system, the importance of the arginine residue at position 53 (R53) in B3 V(H). METHODS: R53 was altered, by site-directed mutagenesis, to serine, asparagine, or lysine, to create 3 expressed variants of V(H). In addition, the germline sequence of the V(H)3-23 gene (from which B3 V(H) is derived) was expressed either with or without arginine at position 53. These 5 new heavy chains, as well as wild-type B3 V(H), were expressed with 4 different light chains, and the resulting antibodies were assessed for their ability to bind to nucleosomes, alpha-actinin, cardiolipin, ovalbumin, beta(2)-glycoprotein I (beta(2)GPI), and the N-terminal domain of beta(2)GPI (domain I), using direct binding assays. RESULTS: The presence of R53 was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of R53 reduced binding to alpha-actinin, ovalbumin, beta(2)GPI, and domain I of beta(2)GPI. The combination B3 (R53S) V(H)/B3 V(L) bound human, but not bovine, beta(2)GPI. CONCLUSION: The fact that the R53S substitution significantly alters binding of B3 to different clinically relevant antigens, but that the alteration is in opposite directions depending on the antigen, implies that this arginine residue plays a critical role in the affinity maturation of antibody B3.     

Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito

During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.