Induction of platelet thrombi by bacteria and antibodies

We have characterized 2 distinct mechanisms through which infectious agents may promote platelet adhesion and thrombus formation in flowing blood, thus contributing to the progression of disease. In one case, the process initiates when the integrin alpha(IIb)beta(3) mediates platelet arrest onto immobilized bacterial constituents that have bound plasma fibrinogen. If blood contains antibodies against the bacteria, immunoglobulin (Ig) G may cluster on the same surface and activate adherent platelets through the Fc(gamma)RIIA receptor, leading to thrombus growth. As an alternative, bacteria that cannot bind fibrinogen may attach to substrates, such as immobilized plasma proteins or components of the extracellular matrix, which also support platelet adhesion. As a result of this colocalization, IgG bound to bacteria can activate neighboring platelets and induce thrombus growth regardless of their ability to initiate platelet-surface contact. Our results demonstrate that intrinsic constituents of infectious agents and host proteins play distinct but complementary roles in recruiting platelets into thrombi, possibly contributing to complications of acute and chronic infections.     

The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.     

The tetraspanin superfamily member CD151 regulates outside-in integrin alphaIIbbeta3 signaling and platelet function

The tetraspanin family member CD151 forms complexes with integrins and regulates cell adhesion and migration. While CD151 is highly expressed in megakaryocytes and to a lesser extent in platelets, its physiologic role in platelets is unclear. In this study, we investigate the physical and functional importance of CD151 in murine platelets. Immunoprecipitation/Western blot studies reveal a constitutive physical association of CD151 with integrin alpha(IIb)beta(3) complex under strong detergent conditions. Using CD151-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta(3) signaling with defective platelet aggregation responses to protease-activated receptor 4 (PAR-4) agonist peptide, collagen, and adenosine diphosphate (ADP); impaired platelet spreading on fibrinogen; and delayed kinetics of clot retraction in vitro. This functional integrin alpha(IIb)beta(3) defect could not be attributed to altered expression of integrin alpha(IIb)beta(3). CD151(-/-) platelets displayed normal platelet alpha granule secretion, dense granule secretion, and static platelet adhesion. In addition, CD151(-/-) platelets displayed normal "inside-out" integrin alpha(IIb)beta(3) signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol 1,4,5 triphosphate (IP(3)) levels. This study provides the first direct evidence that CD151 is essential for normal platelet function and that disruption of CD151 induced a moderate outside-in integrin alpha(IIb)beta(3) signaling defect.     

Calpain regulation of integrin alpha IIb beta 3 signaling in human platelets

Efficient platelet adhesion and aggregation at sites of vascular injury requires the synergistic contribution of multiple adhesion receptors. The initial adhesion of platelets to subendothelial matrix proteins involves GPIb/V/IX and one or more platelet integrins, including integrin alpha IIb beta 3, alpha 2 beta 1, alpha 5 beta 1 and possibly alpha 6 beta 1. In contrast, platelet-platelet adhesion (platelet cohesion or aggregation) is mediated exclusively by GPIb/V/IX and integrin alpha IIb beta 3. Integrin alpha IIb beta 3 is a remarkable receptor that not only stabilizes platelet-vessel wall and platelet-platelet adhesion contacts, but also transduces signals necessary for a range of other functional responses. These signals are linked to cytoskeletal reorganization and platelet spreading, membrane vesiculation and fibrin clot formation, and tension development on a fibrin clot leading to clot retraction. This diverse functional role of integrin alpha IIb beta 3 is reflected by its ability to induce the activation of a broad range of signaling enzymes that are involved in membrane phospholipid metabolism, protein phosphorylation, calcium mobilization and activation of small GTPases. An important calcium-dependent signaling enzyme involved in integrin alpha IIb beta 3 outside-in signaling is the thiol protease, calpain. This enzyme proteolyses a number of key structural and signaling proteins involved in cytoskeletal remodeling and platelet activation. These proteolytic events appear to play a potentially important role in modulating the adhesive and signaling function of integrin alpha IIb beta 3.     

Impaired "outside-in" integrin alphaIIbbeta3 signaling and thrombus stability in TSSC6-deficient mice

We investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin alpha(IIb)beta(3) complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin alpha(IIb)beta(3) expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal "inside-out" integrin alpha(IIb)beta(3) signaling (fluorescein isothiocyanate [FITC]-fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin alpha(IIb)beta(3) "outside-in" signaling events in platelets and is necessary for stability of arterial thrombi in vivo.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.     

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.     

Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets

When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.     

Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets

Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by alpha(IIb)beta(3) integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0. 02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to alpha(IIb)beta(3) integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab')(2) fragments of a monoclonal antibody specific for alpha(IIb)beta(3), demonstrating that, like ERK2, alpha(IIb)beta(3) integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of alpha(IIb)beta(3) integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet alpha(IIb)beta(3). The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by alpha(IIb)beta(3) engagement and positively by mechanical forces in platelets.     

Platelet HDL(3) binding sites are not related to integrin alpha(IIb)beta(3) (GPIIb-IIIa)

Early studies considered that fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha(IIb)beta(3)) is the binding site for low-density lipoprotein (LDL) and high-density lipoprotein type 3 (HDL(3)). Recent data, however, do not support the hypothesis that the binding of LDL to human intact resting platelets is related to integrin alpha(IIb)beta(3). In this study we present evidence that platelet integrin alpha(IIb)beta(3) is also not involved in the interaction of HDL(3) and human intact resting platelets. Firstly, specific ligands for platelet integrin alpha(IIb)beta(3), such as fibrinogen, vitronectin, von Willebrand factor and fibronectin, were unable to inhibit the binding of HDL(3) to intact resting platelets. Secondly, the HDL(3) binding characteristics (K(d) and B(max) values), the activation of protein kinase C (PKC) and the inhibition of thrombin-induced inositoltriphosphate (IP(3)) formation and calcium (Ca(2+)) mobilization mediated by HDL(3) particles were similar in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, which are characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. In contrast, nitrosylation of tyrosine residues of HDL(3) by tetranitromethane fully abolished both the ability of particles to interact with its specific binding sites and the functional effects. Thirdly, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Bak(a/B) and anti-PL(A1/2)), anti-integrin subunits (anti-alpha(V) and anti-beta(3)), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, M8 and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect the binding of HDL(3) particles to human intact resting platelets. Overall results show that neither the GPIIb-IIIa complex nor GPIIb or GPIIIa individually are the membrane binding proteins for HDL(3)on intact resting platelets.     

Internalization of bound fibrinogen modulates platelet aggregation

In agonist-stimulated platelets, the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) is converted from an inactive to an active fibrinogen receptor, thereby mediating platelet aggregation. With time after agonist addition, at least two events occur: fibrinogen becomes irreversibly bound to the platelet and, when stirring is delayed, platelets lose the ability to aggregate despite the presence of maximally bound fibrinogen. Because we previously identified an actively internalized pool of alpha IIb, beta 3 in platelets, we explored the possibility that both of these events might result from the internalization of fibrinogen bound to active alpha IIb beta 3. Under conditions of irreversible fibrinogen binding, fluorescence microscopy showed that biotinylated fibrinogen is rapidly internalized by activated platelets to a surface-inaccessible, intracellular pool. Flow cytometric analysis showed that the observed loss in accessibility to extracellular probes immediately precedes a loss in ability to the platelets to aggregate. Moreover, prevention of irreversible fibrinogen binding results in a prevention of internalization and a retention of aggregation capacity. Thus, the internalization of fibrinogen from the activated platelet surface appears to contribute not only to the irreversible phase of fibrinogen binding, but also to the downregulation of platelet adhesiveness. Fibrinogen internalization is therefore likely to represent a fundamental regulatory mechanism that modulates platelet function.     

The small proteoglycan decorin supports adhesion and activation of human platelets

Decorin is a small leucine-rich proteoglycan able to interact with several molecules of the subendothelial matrix, such as collagen and fibronectin. In this work, we investigated the ability of purified decorin to support adhesion of human platelets. We found that gel-filtered platelets were actually able to interact with immobilized decorin. Platelet adhesion to decorin was time dependent, required the presence of Mg(2+) ions, and was totally mediated by the protein core of the proteoglycan. Platelet stimulation with either adenosine diphosphate (ADP) or a thrombin receptor-activating peptide significantly increased interaction of these cells with the proteoglycan. Upon adhesion to immobilized decorin a number of platelet proteins were found to become tyrosine-phosphorylated. By immunoprecipitation experiments with specific antibodies, the tyrosine phosphorylation of the tyrosine kinase Syk and the phospholipase Cgamma2 (PLCgamma2) isozyme was demonstrated in decorin-adherent platelets. Interaction of platelets with decorin was selectively prevented by 2 different antibodies against membrane integrin alpha(2)beta(1), but not by a number of antibodies against other membrane receptors. In addition, integrin alpha(2)beta(1), purified from platelet membranes, was able to specifically interact with immobilized decorin. Finally, purified decorin bound to Sepharose beads could precipitate integrin alpha(2)beta(1) from a platelet membrane glycoprotein preparation. Therefore, these results demonstrate that human platelets can bind to immobilized decorin through integrin alpha(2)beta(1), and that this interaction results in the tyrosine phosphorylation of intracellular proteins.     

Activation of alphaIIbbeta3 is a sufficient but also an imperative prerequisite for activation of alpha2beta1 on platelets

Platelet integrins alpha2beta1 and alphaIIbbeta3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin alphaIIbbeta3 is not only sufficient, but also a prerequisite for alpha2beta1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of alpha2beta1, as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of alphaIIbbeta3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful alpha2beta1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of alphaIIbbeta3 for proper alpha2beta1 activation on platelets was demonstrated by using the alphaIIbbeta3 antagonist aggrastat, which was able to completely abolish alpha2beta1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking alphaIIbbeta3 confirmed the indispensability of alphaIIbbeta3 for alpha2beta1 activation.     

Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin

Thrombopoietin (TPO) is a recently identified growth factor that regulates megakaryocytopoiesis. Its receptor, c-Mpl, is expressed in megakaryocyte progenitors, mature megakaryocytes, and human blood platelets. We have observed that TPO treatment of human platelets resulted in tyrosine phosphorylation of several cellular proteins, including the c-Mpl receptor and the 85-kD subunit of phosphatidylinositol 3-kinase (PI3-K). TPO stimulated this tyrosine phosphorylation in a time-dependent manner, reaching a maximum in 5 minutes. The tyrosine phosphorylation of PI 3-K was dependent on the concentration of TPO and reached a maximum at concentrations between 50 and 100 ng/mL. This phosphorylation was independent of extracellular fibrinogen and ligation of the alpha IIb beta 3 integrin. In contrast, TPO, in the presence of exogenous fibrinogen, induced concentration- dependent platelet aggregation, which was blocked by the soluble c-Mpl receptor. Increasing TPO concentrations modulated the degree of the primary wave of aggregation and the lag phase, but not the slope or maximum of the secondary wave of aggregation. This secondary aggregation was controlled by the addition of apyrase, suggesting an adenosine diphosphate (ADP)-dependent mechanism. Treatment of platelets with TPO resulted in augmented binding of 125I-fibrinogen to intact platelets, with a 50% effect (EC50) occurring between 5 and 10 ng/mL. TPO-induced binding of fibrinogen to platelets was comparable in degree with that observed by stimulation with 10 mumol/L ADP. In an immobilized collagen-platelet adhesion assay, a significant increase in the attachment of TPO-stimulated platelets was observed. This effect was dependent on the concentration of TPO. At 50 ng/mL of TPO, platelet attachment to collagen increased threefold compared with the buffer control. Furthermore, the presence of fibrinogen did not significantly alter TPO augmentation of the platelet-collagen interaction. This interaction was mediated by the Arg-Gly-Asp (RGD) adhesion recognition sequence, as it was completely abolished by 100 mumol/L of the RGDS peptide. A fraction of the TPO-dependent platelet attachment to a collagen-coated surface was insensitive to treatment with prostaglandin E1. Furthermore, antibody to alpha IIb integrin partially inhibited platelet attachment to collagen, suggesting that the integrin alpha IIb beta 3 participates in this association. These data indicate that TPO might function not only as a cytokine in megakaryocyte growth and differentiation, but may also participate in direct platelet activation and modulate platelet-extracellular matrix interactions.     

Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking.

To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi-Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor-mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.     

Integrin alpha 4 beta 1 and glycoprotein IV (CD36) are expressed on circulating reticulocytes in sickle cell anemia

The abnormal adherence of red blood cells, especially circulating reticulocytes (erythrocyte precursors), to the endothelium is believed to contribute to vascular occlusion observed in patients with sickle cell disease. Although several plasma proteins including von Willebrand factor and fibronectin have been proposed to mediate this adhesion, the mechanism of sickle cell adhesion to the endothelium remains unknown. Using flow cytometry, we screened sickle red blood cells with monoclonal antibodies (MoAbs) against known adhesion receptors and detected integrin subunits alpha 4 and beta 1 and the nonintegrin glycoprotein IV on reticulocytes but not on erythrocytes. No reactivity was detected against integrin subunits alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 2, beta 3, integrin alpha IIb beta 3, or the nonintegrin glycoprotein Ib. Immunoprecipitation of reticulocytes with either alpha 4- or beta 1-specific antibodies identified the alpha 4 beta 1 complex (alpha 4(70) and alpha 4(80) forms), a receptor for fibronectin and vascular cell adhesion molecule-1. An antibody against glycoprotein IV, a receptor reported to bind thrombospondin and collagen, immunoprecipitated an 88-kD protein consistent with its reported M(r). MoAbs against alpha 4 and glycoprotein IV bound to an average of 4,600 and 17,500 sites per reticulocyte, respectively. Identification of alpha 4 beta 1 and glycoprotein IV on reticulocytes suggests both plasma-dependent and independent mechanisms of reticulocyte adhesion to endothelium and exposed extracellular matrix.     

Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3).

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin alpha(IIb)beta(3). GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin alpha(IIb)beta(3) promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin alpha(IIb)beta(3), particularly under shear, have remained poorly defined. In this study, the potential involvement of type 1 phosphoinositide (PI) 3-kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3-kinase lipid product, PtdIns(3,4)P(2). Under static conditions, inhibiting PI 3-kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin alpha(IIb)beta(3) activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3-kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Real-time analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3-kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3-kinase dramatically reduced intracellular calcium mobilization and integrin alpha(IIb)beta(3) activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3-kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3-kinase-dependent and -independent mechanisms, whereas under shear PI 3-kinase is indispensable for VWF-induced calcium release.     

Vascular endothelial growth factor-stimulated endothelial cells promote adhesion and activation of platelets

Coagulation abnormalities, including an increased platelet turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested the hypothesis that platelets contribute to angiogenesis. Stimulation with vascular endothelial growth factor (VEGF, 25 ng/mL) of human umbilical vein endothelial cells (HUVECs) promoted adhesion of nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs with basic fibroblast growth factor (bFGF) did not promote platelet adhesion. By blocking tissue factor (TF) activity, platelet adhesion was prevented and antibodies against fibrin(ogen) and the platelet-specific integrin, alpha(IIb)beta(3), inhibited platelet adhesion for 70% to 90%. These results indicate that VEGF-induced platelet adhesion to endothelial cells is dependent on activation of TF. The involvement of fibrin(ogen) and the alpha(IIb)beta(3) integrin, which exposes a high-affinity binding site for fibrin(ogen) on platelet activation, indicates that these adhering platelets are activated. This was supported by the finding that the activity of thrombin, a product of TF-activated coagulation and a potent platelet activator, was required for platelet adhesion. Finally, platelets at physiologic concentrations stimulated proliferation of HUVECs, indicative of proangiogenic activity in vivo. These results support the hypothesis that platelets contribute to tumor-induced angiogenesis. In addition, they may explain the clinical observation of an increased platelet turnover in cancer patients. Platelets may also play an important role in other angiogenesis-dependent diseases in which VEGF is involved, such as diabetes and autoimmune diseases. (Blood. 2000;96:4216-4221)     

Variability of integrin alpha 2 beta 1 activity on human platelets

The activity and surface antigenicity of alpha 2 beta 1 on platelets from 27 normal subjects were found to vary significantly. A fourfold range of surface antigen correlates with a 20-fold variation in the ability of nonactivated, washed platelets to adhere to type I collagen and a fivefold variation in the adhesion of platelets to type III collagen. These differences in surface receptor are reflected in significant variation in the lag time required for type I collagen- induced platelet aggregation in platelet-rich plasma. Among the same individuals, no difference was observed in surface levels or activities of two other platelet integrins, the fibronectin receptor alpha 5 beta 1 and the fibrinogen receptor alpha IIb beta 3. In all cases studied, we observed complimentary differences in the incorporation of 125I into surface alpha 2 beta 1, in quantity of surface alpha 2 beta 1 antigens, and in alpha 2 beta 1 collagen receptor activity. Despite variations in these parameters, there was no difference in the electrophoretic mobility or isoelectric point of either integrin subunit among the individuals studied. The wide range of activity and antigenicity of this platelet collagen receptor may result from polymorphism(s) in the alpha 2 beta 1 genes, or the activity of alpha 2 beta 1 may be variably regulated by another gene product. The heterogeneity of platelet alpha 2 beta 1 that we describe in this report certainly explains previous discrepancies concerning the contributions of this integrin to platelet adhesion to collagens. Most importantly, differences in surface collagen receptor activity may correlate with a long-term risk toward thrombosis, impaired hemostasis, and/or cardiovascular disease.