Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca2+ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP3) and that treating platelets with the IP3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca2+ flux and platelet shape change. Normal shape change was observed in G alpha q-/- mouse platelets, excluding a role for PLC beta isoforms in this process. However, decreased shape change and Ca2+ mobilization were observed in mice lacking PLC gamma 2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca2+ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca2+ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-Fc gamma RIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcR gamma chain. Taken together, these studies define an important role for PLC gamma 2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.     

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y₁₂ and thromboxane receptor through secreted ADP and TxA₂, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.     

Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen.

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affinity chromatography. The domain 3-derived peptide, thrombin, HK, factor XII, as well as antibody to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above-mentioned ligands bound to this protein. Isolation of platelet membrane proteins using a monoclonal anti-glycocalicin antibody column revealed the same HK binding protein at 74 kD, which was reactive with anti-GPIb and represents a GPIb fragment. By photoaffinity labeling, HK interacted with membrane GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, (125)I-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest that the GPIb alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3.     

A platelet secretion pathway mediated by cGMP-dependent protein kinase

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.     

A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets.

Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.     

Involvement of platelet glycoprotein Ib in platelet microparticle mediated neutrophil activation

Summary Platelet microparticles (MPs) are membrane vesicles shed by platelets after activation, and carry antigens characteristic of intact platelets, such as glycoprotein (GP) IIb/IIIa, GPIb and P-selectin. Elevated platelet MPs have been observed in many disorders in which platelet activation is documented. Recently, platelet GPIb has been implicated in the mediation of platelet–leukocyte interaction via binding to its ligand Mac-1 on leukocyte. The role of GPIb for mediating adhesion-activation interactions between platelet MPs and leukocytes has not been clarified. In this study we investigate the role of GPIb in the interplay between platelet MPs and neutrophils. Platelet MPs were obtained from collagen-stimulated platelet-rich plasma (PRP). In a study model of neutrophil aggregation, platelet MPs can serve a bridge to support neutrophil aggregation under venous level shear stress, suggesting that platelet MPs may enhance leukocyte aggregation, which would bear clinical relevance in diseases where the platelet MPs are elevated. The level of aggregation can be reduced by GPIb blocking antibodies, AP1 and SZ2, but not by anti-CD18 mAb. The GPIb blocking antibodies also decreased platelet MP-mediated neutrophil activation, including β2 integrin expression, adherence-dependent superoxide release and platelet MP-mediated neutrophil adherence to immobilized fibrinogen. Our data provide the evidence for the involvement of GPIb–Mac-1 interaction in the cross-talk between platelet MPs and neutrophils.     

Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (~15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.     

Activation of platelet concentrate during preparation and storage.

This review will discuss how stored platelets become activated and will examine their ability to function and survive in vivo, posttransfusion. Experimental methods which have been shown to alter platelets during storage will be detailed. Using beta-thromboglobulin (beta-TG) and surface adhesion receptors as markers, investigators have examined the activation changes in platelet concentrates during preparation and storage. Resuspension of the platelet pellet after isolation of platelet-rich plasma appears to play a major role in producing platelet activation and beta-TG release during preparation. However, there is a significant amount of interdonor variability in platelet activation even at this early stage of storage. Over 5 days of storage, platelets release approximately 50% of their beta-TG contents. Furthermore, between 40% and 60% of the platelets express the alpha-granule membrane protein, P-selectin (GMP-140), during storage, which is also indicative of platelet activation. These activation changes correlate to some degree with platelet recovery posttransfusion but clearly do not explain the full lesion of platelet storage. The surface density of two platelet membrane receptors, glycoproteins (GP) Ib and IIb/IIIa, also change with activation, although in opposite directions. Platelet surface GPIb decreases initially with storage and then recovers, perhaps due to its relocation to the platelet surface from an intracellular pool. In contrast to GPIb, mean platelet surface GPIIb/IIIa increases slightly during storage, probably as a consequence of platelet activation and release of alpha-granule GPIIb/IIIa to the surface. Some hypotheses are offered regarding how these activated platelets can continue to circulate after transfusion. Further exploration of the platelet storage lesion will hopefully provide needed answers and thus permit better treatment of hemostatic disorders in the future.     

The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets

A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.     

Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.     

"Thrombin" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides.

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.     

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background

Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. α_IIbβ₃ is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods

Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results

Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions

We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

     

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.     

Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets.

In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G-proteins, or collagen which is known to mobilise a tyrosine kinase-dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin alpha(IIb)beta(3) engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3-kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen.     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献   

Inhibition of thrombin-induced platelet activation by leupeptin. Implications for the participation of calpain in the initiation of platelet activation

Inhibitors of calcium-dependent proteases (calpains) such as leupeptin and antipain have been shown to selectively inhibit platelet activation by thrombin. Based upon this observation, it has been proposed that calpains play a role in the initiation of platelet activation. In the present studies, we have examined the effect of leupeptin on the earliest known event in thrombin-induced platelet activation: the interaction between the agonist, its receptors, and the guanine nucleotide-binding proteins which stimulate phospholipase C (Gp) and inhibit adenylyl cyclase (Gi). We found that leupeptin inhibited thrombin's ability to stimulate phosphoinositide hydrolysis, suppress cAMP formation, and dissociate Gp and Gi into subunits. Leupeptin had no effect, however, on the same responses to other agonists or on thrombin binding to platelets. Although these observations might suggest, as others have concluded, that calpain is involved in the initiation of platelet activation by thrombin, we also found that: 1) substituting platelet membranes for intact platelets and decreasing the free Ca2+ concentration below the threshold required for calpain activation did not diminish the effects of leupeptin on phosphoinositide hydrolysis and cAMP formation, 2) washing the platelets after incubation with leupeptin reversed the effects of the inhibitor, 3) permeabilizing the platelets with saponin did not enhance the inhibitory effects of leupeptin, and 4) leupeptin inhibited the proteolysis of fibrinogen and the hydrolysis of S2238 by thrombin. Similar results in these assays were obtained with antipain. Therefore, our observations suggest that the inhibition of platelet activation by leupeptin is due to a direct interaction with thrombin and need not reflect a role for calpain in the initiation of platelet activation.