A G(i) -independent mechanism mediating Akt phosphorylation in platelets.

BACKGROUND: The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. OBJECTIVES AND METHODS: We used P2Y1 knockout mice to address the role of P2Y12 in Akt phosphorylation in response to thrombin receptors in platelets. RESULTS: Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr³°8 and Ser47³ in P2Y12-deficient platelets. AYPGKF-induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y12 -independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin ß3 deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y12-deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. CONCLUSIONS: Our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.

Evaluation of the physiological significance of botrocetin/ von Willebrand factor in vitro signaling.

BACKGROUND: A signaling pathway is difficult, if not impossible, to elucidate in platelets using only in vivo studies. Likewise, the physiological significance of signaling information obtained exclusively from in vitro observations is unknown. Therefore, both in vitro and in vivo experiments are required to establish the physiological significance of a signaling pathway. OBJECTIVE: To evaluate the physiological significance of signaling data obtained from botrocetin (bt)/von Willebrand factor (VWF)-stimulated washed platelets. METHOD: Stable thrombus formation in response to FeCl(3)-induced injury of the mouse carotid artery was used to evaluate the physiological significance of signaling data obtained from bt/VWF-stimulated washed platelets. RESULTS: Syk, PLCgamma2, Galphaq and P2Y12, but not LAT, were found either to be required for or to affect stable thrombus formation. Prior in vitro studies had demonstrated that LAT is not required for bt/VWF-induced platelet aggregation in the presence of exogenous fibrinogen. These data provide the first demonstration of the in vivo role for these signaling molecules in GPIb-dependent/initiated signal transduction and are consistent with the signaling pathway deduced from in vitro studies of bt/VWF-stimulated washed platelets using metabolic inhibitors and knockout mice. CONCLUSION: The broad agreement between the in vitro and the in vivo results establish that bt/VWF stimulation of washed platelets can provide physiologically significant glycoprotein Ib-dependent/initiated signaling data.

In vivo relevance for platelet glycoprotein Ibalpha residue Tyr276 in thrombus formation.

BACKGROUND: Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the alpha-subunit of GP (GPIbalpha) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIbalpha. OBJECTIVES: This study investigated the in vivo relevance of GPIbalpha residue Tyr276 in hemostasis and thrombosis. METHODS: Transgenic mouse colonies expressing the normal human GPIbalpha subunit or a mutant human GPIbalpha containing a Phe substitution for Tyr276 (hTg(Y276F)) were generated. Both colonies were bred to mice devoid of murine GPIbalpha. RESULTS: Surface-expressed GPIbalpha levels and platelet counts were similar in both colonies. hTg(Y276F) platelets were significantly impaired in binding alpha-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl(3)) demonstrated that hTg(Y276F) mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg(Y276F) animals were also less stable. CONCLUSIONS: The results demonstrate that GPIbalpha residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo.

The roles of ADP and TXA in botrocetin/VWF-induced aggregation of washed platelets.

BACKGROUND: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to alphaIIbbeta3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination-induced GPIbalpha-mediated platelet activation have not been fully described. METHODS: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Galphaq, or alphaIIbbeta3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, alphaIIbbeta3 and Ca2+ in botrocetin/VWF-induced signaling. RESULTS: Our data demonstrate that botrocetin/VWF/GPIbalpha-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbalpha-elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2-induced alphaIIbbeta3 activation and ADP signaling. TXA2 or ADP can activate alphaIIbbeta3, but both are required for alpha-granule secretion and aggregation. Botrocetin/VWF-induced dense granule secretion is Galphaq-dependent. alpha-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an alphaIIbbeta3-dependent manner. CONCLUSIONS: In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates alphaIIbbeta3 requires TXA2. Agglutination-elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes alphaIIbbeta3 activation by a mechanism that is distinct from those used by adhesion, or shear stress-induced GPIb signaling.

AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets.

The peptide LSARLAF (LSA) causes alphaIIbbeta3-dependent platelet activation that results in alpha-granule secretion and aggregation. LSARLAF-induced, alphaIIbbeta3-mediated outside-in signaling causing alpha-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Galphaq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced alphaIIbbeta3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause alpha-granule release by mediating their effects though Galphaq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, alphaIIbbeta3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Galphaq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Galphaq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced alphaIIbbeta3-mediated signaling characterized in this study is alpha-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.

Distinct domains of alphaIIbbeta3 support different aspects of outside-in signal transduction and platelet activation induced by LSARLAF, an alphaIIbbeta3 interacting peptide.

The peptide LSARLAF causes alphaIIbeta3-dependent platelet activation exemplified by secretion, aggregation, spreading and adhesion on fibrinogen, and tyrosine phosphorylation. alphaIIIbeta3-dependent outside-in signal transduction induced by LSARLAF was investigated in variant thrombasthenic platelets which lack most of the cytoplasmic domain of the integrin beta3 subunit (alphaIIbbeta3 delta724). These studies revealed that only certain aspects of this alphaIIbbeta3-dependent outside-in signaling were affected by the beta3 truncation. Specifically, alphaIIbbeta3 delta724 supported LSARLAF-induced platelet aggregation, agglutination and secretion, but failed to trigger cytoskeletal reorganization and platelet spreading on fibrinogen, despite the fact that PMA-induced non alphaIIbbeta3 mediated signaling caused spreading of these platelets on fibrinogen. Thus, distinct domains of alphaIIbbeta3 are required to support different aspects of LSARLAF-induced platelet activation. Furthermore, these studies suggest that not all alphaIIbbeta3-dependent platelet responses require an intact beta3 cytoplasmic tail.

Evidence for new endothelial cell binding sites on fibrinogen.

In vitro assays were used to characterize adhesion of human aortic, microvascular and umbilical vein endothelial cells to various forms of immobilized fibrinogen. All three types of endothelial cells adhered to fibrinogen in a manner that was independent of the Aalpha-chain 572-574 RGD cell binding site. In fact, all three adhered to a fragment of the molecule which is composed of only one D domain (D1) of fibrinogen. A time course study revealed that extensive adhesion of endothelial cells on the ligand coated surface occurred between one and two hours incubation. The anti-fibrinogen gammaA-chain monoclonal antibody 4A5 as well as 4A5 Fabs, blocked adhesion of endothelial cells to fibrinogen, not vitronectin. The inhibitory effects of 4A5 seemed to be indirect because the endothelial cells adhered to the recombinant fibrinogen gamma407 (which lacks the gamma-chain AGDV sequence of the carboxyl terminal 4A5 binding site) as well as they did to normal recombinant fibrinogen. A recombinant fibrinogen lacking the gamma-chain AGDV sequence, containing RGE in place of RGD at the gamma-chain 572-574 and 95-97 positions, also supported endothelial cell adhesion. The anti-alphavbeta3 antibody, LM609, blocked adhesion of endothelial cells to fibrinogen. The peptide GRGDSP inhibited endothelial cell adhesion on fibrinogen and vitronectin. These results demonstrate that alphavbeta3 mediated adhesion (attachment and spreading) of HUVECs to fibrinogen may use a site in the D domain of fibrinogen and is not dependent on the Aalpha-chain RGD (95-97 and 572-574) sequences, as has been shown in shorter term (where cells were rounded) experiments, or the alphaA-chain 408-411 cell binding sites. Thus, the data reveal the existence of another unidentified site(s) on fibrinogen which can support the irreversible adhesion (attachment and spreading) of endothelial cells.

Adhesion of microvascular endothelial cells to metallic implant surfaces.

The objective of this study was to explore the molecular mechanisms of adhesion of endothelial cells (ECs) to implant grades of titanium alloy (Ti) and stainless steel (SS), compared to tissue culture polystyrene (PS). The idea is that promotion of EC adhesion to implant surfaces during the initial stages of healing may be critical in the formation of a capillary bed intimately associated with the implant surface. Ultimately this could be expected in turn to promote bone formation close to the surface and a more stable implant/bone interface. Surfaces were coated with either peak 1 fibrinogen gammaAgammaA, fibrinogen Fr I-9, fibrinogen fragment D1, fibronectin, vitronectin, or fetal calf serum and then post-coated with bovine serum albumin (BSA) to block non-specific cell adhesion. Surfaces with BSA alone and no other protein coating were also evaluated. Fibronectin coating maximized cell adhesion on all three surfaces, and adhesion was highest on PS. BSA blocked cell adhesion to PS (and most adhesion to SS) much better than to Ti. These results provide evidence that BSA adsorption on the metal surface is unable to effectively block the adhesion of the cells to the Ti. These data may provide a basis for understanding in vivo observations that soft tissue becomes attached to a Ti surface more rapidly and with more bone formation than to SS. Evidence is also presented that alphavbeta3 plays an important role in adhesion of ECs to the Ti surface. These experiments also provide preliminary data which may reflect some of the features of initial EC adhesion to metal implants.

A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3.

Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)

Peptide LSARLAF activates alpha(IIb)beta3 on resting platelets and causes resting platelet aggregate formation without platelet shape change.

Adhesion of resting platelets to fibrinogen was enhanced by a peptide which was designed to bind near the presumptive fibrinogen gamma-chain binding site of the alpha subunit of the integrin alpha(IIb)beta3. This peptide, but not a scrambled control peptide, induced adhesion of resting platelets to fibronectin, vitronectin, von Willebrand factor, and monovalent (lacks one functional gamma-chain) fibrinogen. Resting platelets not treated with the agonist peptide did not adhere to these ligands. Agonist peptide induced adhesion of resting platelets to Fg was not secretion dependent and was inhibited by the monoclonal antibody 7E3. The agonist peptide caused aggregation of resting platelets on resting platelets adherent to immobilized Fg without causing platelet shape change. Therefore, the agonist peptide may activate alpha(IIb)beta3 by directly inducing a conformation change in the receptor on resting platelets.

The role of putative fibrinogen Aalpha-, Bbeta-, and GammaA-chain integrin binding sites in endothelial cell-mediated clot retraction.

In this study, endothelial cell-mediated clot retraction was supported by fibrin generated from several purified fractions of plasma fibrinogen, purified proteolytic fragments of plasma fibrinogen, recombinant normal fibrinogen, and recombinant variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin receptors that support clot retraction. Specifically, fibrinogens lacking Aalpha-chain RGD residues at 572-574 or lacking the gamma-chain residues AGDV 408-411 supported endothelial cell-mediated clot retraction as well as intact fibrinogen. Thus, clot retraction mediated by endothelial cells is not dependent on either of these sites. A variety of monoclonal antibodies against the integrin alphavbeta3 partially inhibited the endothelial cell-mediated retraction of clots formed from plasma fibrinogen. As expected, an antibody to the platelet integrin alphaIIbbeta3 did not inhibit endothelial cell-mediated clot retraction. These results indicate that this retraction is mediated at least in part by alphavbeta3. These results support the conclusion that (a) neither of the two fibrinogen cell binding sites described above is required to support clot retraction or that (b) either site alone or in conjunction with other fibrin(ogen) region(s) can support clot retraction. Thus, endothelial cell-mediated clot retraction appears to be dependent on fibrinogen cell binding sites other than those required to support adhesion of resting platelets to immobilized fibrinogen and platelet aggregation.

The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin alphaIIbbeta3.

A novel peptide (designed to bind to alphaIIbbeta3) caused platelet aggregation and aggregation-independent secretion of the contents of alpha-granules in an alphaIIbbeta3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, alphaIIbbeta3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized alphaIIbbeta3. The agonist peptide apparently activates alphaIIbbeta3 by directly inducing a conformational change in the receptor. This change activates the platelets and causes secretion in a manner independent of fibrinogen binding.

Variabilin, a novel RGD-containing antagonist of glycoprotein IIb-IIIa and platelet aggregation inhibitor from the hard tick Dermacentor variabilis.

A novel inhibitor of human platelet aggregation, named variabilin, was isolated from salivary glands of the hard tick Dermacentor variabilis using a combination of gel filtration and high pressure liquid chromatography. Variabilin was a potent antagonist of the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa; alphaIIbbeta3) and the vitronectin receptor alphavbeta3. Amino acid sequence analysis by Edman degradation revealed that it has 47 residues, with a molecular weight of 4968.5. Like many other naturally occurring antagonists of GPIIb-IIIa, variabilin contains the RGD (Arg-Gly-Asp) motif. However, unlike the RGD-containing antagonists of GPIIb-IIIa, the RGD sequence of variabilin is not positioned in a loop bracketed by cysteine residues. It has little sequence homology to the other known naturally occurring antagonists of GPIIb-IIIa, including the disintegrins from snakes, decorsin and ornatin from leeches, and disagregin from soft ticks. Variabilin is the first RGD-containing antagonist isolated from ticks.

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation of Rana pipiens.

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frog Rana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373-1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124-131 of beta 1 subunit of a variety of integrins including alpha 5 beta 1. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).

Characterization of adhesion of non-exogenously stimulated and resting platelets in normal plasma to fibrinogen and its fragments.

Platelet adhesion to various forms of fibrinogen was studied using platelets in plasma and washed platelets. The study was designed to determine if platelets prepared with minimal handling in plasma at physiological pH containing normal levels of Ca2+ have different requirements for adhesion to immobilized fibrinogen than do washed platelets tested in the absence of plasma. Exposure of platelets to citrate and low pH did not seem to affect the requirements of washed platelets for adhesion to fibrinogen. Nonetheless, behavioural differences between these two types of platelets were seen. Surprisingly, in the absence of exogenous activation normal platelets in plasma behaved qualitatively as stimulated washed platelets. That is, both types of platelets adhered to all forms of fibrinogen which possessed at least one gamma-chain carboxyl terminal platelet binding site. Platelets in plasma treated with prostaglandin E1 (resting platelets) adhered only to forms of fibrinogen which contained two gamma-chain platelet binding sites. These observations also demonstrate that the fibrinogen alpha-chain arginine-glycine-aspartic acid-phenylalanine and arginine-glycine-aspartic acid-serine sequences are not necessary or sufficient to mediate the adhesion of resting or stimulated platelets in plasma to fibrinogen. The presence of endogenous adenosine diphosphate appears to account, at least in part, for the ability of normal platelets in plasma to adhere to forms of fibrinogen which have only one gamma-chain platelet binding site.

Complementary peptides that interfere with platelet aggregation and adherence.

This article describes the application of the molecular recognition hypothesis to the critically important process of fibrinogen binding to platelets, a process that is the subject of extensive and intensive basic and clinical research. The objectives of the studies summarized below were to design, synthesize, and characterize peptides that can inhibit the binding of fibrinogen and related ligands to human platelets and thereby prevent platelet aggregation, adhesion, and clot retraction. The purpose of doing this work was twofold: first, to determine whether the molecular recognition hypothesis could serve as a useful rationale for the design of peptides that can specifically inhibit the binding of fibrinogen and related ligands to platelets; and second, to use these peptides to try to learn where fibrinogen binds to the platelet fibrinogen receptor. It was hoped that the results obtained not only would provide insight into platelet function but also might provide a rationale for the design of a clinically useful anti-thrombotic agent. Although our studies are not complete, they have resulted in the design of a variety of peptides that can inhibit platelet aggregation, adhesion, and clot retraction as a consequence of specifically inhibiting the binding of fibrinogen and related ligands to the platelet fibrinogen receptor. Although none of these peptides appears to be ligand specific, one or two of them may be specific for platelets.