Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha IIb beta 3 adhesive function in platelets

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110 beta and p110 gamma) have been implicated in G(i)-dependent integrin alpha(IIb)beta(3) regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110 beta is primarily responsible for G(i)-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin alpha(IIb)beta(3) activation in p110 gamma(-/-) platelets was not associated with alterations in the levels of PI(3,4)P(2) or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110 gamma principally regulated integrin alpha(IIb)beta(3) activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110 gamma, led to a major reduction in integrin alpha(IIb)beta(3) activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110 gamma in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110 beta and p110 gamma is critical for P2Y(12)/G(i)-dependent integrin alpha(IIb)beta(3) regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.     

RhoA sustains integrin alpha IIbbeta 3 adhesion contacts under high shear

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, alpha(IIb)beta(3). Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin alpha(IIb)beta(3), by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin alpha(IIb)beta(3)-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase had no effect on integrin alpha(IIb)beta(3) activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin alpha(IIb)beta(3) adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin alpha(IIb)beta(3) adhesion contacts under conditions of high shear stress.     

Platelet factor XIII and calpain negatively regulate integrin alphaIIbbeta3 adhesive function and thrombus growth

Excessive accumulation of platelets at sites of athero-sclerotic plaque rupture leads to the development of arterial thrombi, precipitating clinical events such as the acute coronary syndromes and ischemic stroke. The major platelet adhesion receptor glycoprotein (GP) IIb-IIIa (integrin alpha(IIb)beta3) plays a central role in this process by promoting platelet aggregation and thrombus formation. We demonstrate here a novel mechanism down-regulating integrin alpha(IIb)beta3 adhesive function, involving platelet factor XIII (FXIII) and calpain, which serves to limit platelet aggregate formation and thrombus growth. This mechanism principally occurs in collagen-adherent platelets and is induced by prolonged elevations in cytosolic calcium, leading to dramatic changes in platelet morphology (membrane contraction, fragmentation, and microvesiculation) and a specific reduction in integrin alpha(IIb)beta3 adhesive function. Adhesion receptor signal transduction plays a major role in the process by sustaining cytosolic calcium flux necessary for calpain and FXIII activation. Analysis of thrombus formation on a type I fibrillar collagen substrate revealed an important role for FXIII and calpain in limiting platelet recruitment into developing aggregates, thereby leading to reduced thrombus formation. These studies define a previously unidentified role for platelet FXIII and calpain in regulating integrin alpha(IIb)beta3 adhesive function. Moreover, they demonstrate the existence of an autoregulatory feedback mechanism that serves to limit excessive platelet accumulation on highly reactive thrombogenic surfaces.     

Importance of temporal flow gradients and integrin alphaIIbbeta3 mechanotransduction for shear activation of platelets

Disturbances of blood flow play an important role in promoting platelet activation and arterial thrombus formation in stenosed, injured, atherosclerotic arteries. To date, glycoprotein Ib (GPIb) has been considered the primary platelet mechanosensory receptor, responding to increased shear with enhanced adhesive and signaling function. We demonstrate here that von Willebrand factor-GPIb interaction is inefficient at inducing platelet activation even when platelets are exposed to very high wall shear stresses (60 dyn/cm(2)). Rapid platelet activation under flow was only observed under experimental conditions in which transiently adherent platelets were exposed to sudden accelerations in blood flow. Platelet responsiveness to temporal shear gradients was integrin alpha(IIb)beta(3)-dependent and occurred only on a von Willebrand factor substrate, as platelets forming integrin alpha(IIb)beta(3) adhesive contacts with immobilized fibrinogen were unresponsive to sudden increases in shear. The calcium response induced by temporal shear gradients was distinct from previously identified integrin alpha(IIb)beta(3) calcium responses in terms of its transient nature, its requirement for platelet co-stimulation by the P2Y(1) purinergic ADP receptor, and its dependence on the influx of extracellular calcium. Our studies demonstrate a key role for temporal shear gradients in promoting platelet activation. Moreover, they define for the first time the involvement of P2Y receptors in integrin mechanotransduction.     

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling

Integrin alpha(IIb)beta(3) activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin alpha(IIb)beta(3) signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin alpha(IIb)beta(3) in resting platelets and in human embryonal kidney 293 cells expressing alpha(IIb)beta(3). The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin alpha(IIb) is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to alpha(IIb)beta(3) during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac(alpha) in 293 cells decreased alpha(IIb)beta(3)-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac(alpha) expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated alpha(IIb)beta(3) adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac (alpha)-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A(calpha) expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac (alpha) can negatively regulate integrin alpha(IIb)beta(3) signaling by suppressing the ERK1/2 signaling pathway.     

Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.     

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.     

Proteolysis leads to the appearance of the long form of beta3-endonexin in human platelets

After vessel injury, platelets adhere to the subendothelial matrix. Platelet adhesion leads to activation of the platelet integrin alpha(IIb)beta3, which then binds to fibrinogen, leading to platelet aggregation. It has been shown that a beta3-integrin binding protein, beta3-endonexin, can activate the integrin alpha(IIb)beta3 expressed in transfected CHO cells. Several isoforms of beta3-endonexin are known but it is not clear which isoforms are expressed in platelets and what role they may play during haemostasis. Here, we show that the long form of beta3-endonexin (EN-L) can be detected in platelet lysates several hours after thrombus formation, after long-term storage of platelets and after glucose deprivation. After subcellular fractionation, EN-L is found in the detergent insoluble fraction suggesting that it might be associated with the cytoskeleton. EN-L generation is temperature and Ca++ dependent and requires physiological salt concentrations. Proteolysis is responsible for the appearance of EN-L since a calpain inhibitor prevents its formation and the addition of calpain to platelet lysates induces its formation. The appearance of EN-L seems to be linked to apoptotic events occurring during long-term storage of platelets and, possibly, during late steps of haemostasis after thrombus formation.     

Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets ans transfected Chinese hamster ovary cells

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin alpha(IIb)beta(3). These include the following: 1) examining the sufficiency of GPIb/IX and integrin alpha(IIb)beta(3) to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin alpha(IIb)beta(3) activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin alpha(IIb)beta(3), we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin alpha(IIb)beta(3) mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin alpha(IIb)beta(3). Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150-1800 s(-)(1)). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin alpha(IIb)beta(3) activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin alpha(IIb)beta(3) activation.     

Distinct but critical roles for integrin alphaIIbbeta3 in platelet lamellipodia formation on fibrinogen, collagen-related peptide and thrombin

Integrins are the major receptor type known to facilitate cell adhesion and lamellipodia formation on extracellular matrix proteins. However, collagen-related peptide and thrombin have recently been shown to mediate platelet lamellipodia formation when presented as immobilized surfaces. The aims of this study were to establish if there exists a role for the platelet integrin alpha(IIb)beta(3) in this response; and if so, whether signalling from the integrin is required for lamellipodia formation on these surfaces. Real-time analysis was used to compare platelet morphological changes on surfaces of fibrinogen, collagen-related peptide or thrombin in the presence of various pharmacological inhibitors and platelets from 'knockout' mice. We demonstrate that collagen-related peptide and thrombin stimulate distinct patterns of platelet lamellipodia formation and elevation of intracellular Ca(2+) to that induced by the integrin alpha(IIb)beta(3) ligand, fibrinogen. Nevertheless, lamellipodia formation on collagen-related peptide and thrombin is dependent upon engagement of alpha(IIb)beta(3), consistent with release of alpha(IIb)beta(3) ligand(s) from platelet granules. However, the requirement for signalling by the integrin on fibrinogen can be bypassed by the addition of thrombin to the solution. These observations reveal a critical role for alpha(IIb)beta(3) in forming lamellipodia on collagen-related peptide and thrombin which is dependent on its ability to function as an adhesive receptor but not necessarily on its ability to signal. These results suggest that integrins may play an important role in lamellipodia formation triggered by nonintegrin ligands in platelets and possibly in other cell types.     

Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.     

Phosphoinositide 3-Kinase p110�� Regulates Integrin ��IIb��3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin α_IIbβ₃, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin α_IIbβ₃ activation (affinity modulation) primarily occurs downstream of G_i-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin α_IIbβ₃ bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin α_IIbβ₃ activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin α_IIbβ₃ association with the contractile cytoskeleton. Analysis of integrin α_IIbβ₃ adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin α_IIbβ₃ bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin α_IIbβ₃ receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors.     

Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.     

A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.     

Resistance to thromboembolism in PI3Kgamma-deficient mice.

Platelet aggregation and subsequent thrombosis are the major cause of ischemic diseases such as heart attack and stroke. ADP, acting via G protein-coupled receptors (GPCRs), is an important signal in thrombus formation and involves activation of phosphoinositide 3-kinases (PI3K). When platelets from mice lacking the G protein-activated PI3Kgamma isoform were stimulated with ADP, aggregation was impaired. Collagen or thrombin, however, evoked a normal response. ADP stimulation of PI3Kgamma-deficient platelets resulted in decreased PKB/Akt phosphorylation and alpha(IIb)beta(3) fibrinogen receptor activation. These effects did not influence bleeding time but protected PI3Kgamma-null mice from death caused by ADP-induced platelet-dependent thromboembolic vascular occlusion. This result demonstrates an unsuspected, well-defined role for PI3Kgamma downstream of ADP and suggests that pharmacological targeting of PI3Kgamma has a potential use as antithrombotic therapy.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

Probing chemical and conformational differences in the resting and active conformers of platelet integrin alpha(IIb)beta(3)

Integrin alpha(IIb)beta(3) is the fibrinogen receptor that mediates platelet adhesion and aggregation. The ligand binding function of alpha(IIb)beta(3) is "activated" on the platelet surface by physiologic stimuli. Two forms of alpha(IIb)beta(3) can be purified from platelet lysates. These forms are facsimiles of the resting (Activation State-1 or AS-1) and the active (Activation State-2 or AS-2) conformations of the integrin found on the platelet surface. Here, the differences between purified AS-1 and AS-2 were examined to gain insight into the mechanism of activation. Four major findings are put forth. 1) The association rate (k(1)) between fibrinogen and the integrin is a key difference between AS-1 and AS-2. 2) Although the divalent ion Mn(2+) enhances the ligand binding function of AS-1, this ion is unable to convert AS-1 to AS-2. Therefore, its effect on integrin is unrelated to activation. 3) Peptide mass fingerprints indicate that the chemical structure of AS-1 and AS-2 are virtually identical, calling into question the idea that post-translational modifications are necessary for activation. 4) The two forms of alpha(IIb)beta(3) have significant conformational differences at three positions. These include the junction of the heavy and light chain of alpha(IIb), the divalent ion binding sites on alpha(IIb), and at a disulfide-bonded knot linking the amino terminus of beta(3) to the cysteine-rich domain. These observations indicate that integrin is activated by a series of specific conformational rearrangements in the ectodomain that increase the rate of ligand association.     

Integrin-independent tyrosine phosphorylation of p125(fak) in human platelets stimulated by collagen

Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125(fak) (FAK), in human platelets. An integrin alpha(2)beta(1)-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha(2) and beta(1) integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha(IIb)beta(3). The intracellular Ca(2+) chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha(2)beta(1) is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha(IIb)beta(3) is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca(2+) and protein kinase C activity are essential intermediaries of FAK activation.     

The leech product saratin is a potent inhibitor of platelet integrin alpha2beta1 and von Willebrand factor binding to collagen

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and alpha2beta1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet-collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking alpha2beta1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated alpha2beta1-dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet-rich plasma adhesion to fibrillar collagen, a process dependent upon both alpha2beta1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the alpha2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and alpha2beta1. These results demonstrate that saratin interferes with integrin alpha2beta1 binding to collagen in addition to inhibiting von Willebrand factor-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.