Thrombin-induced Platelet Shape Change Research Articles

CGMP signaling inhibits platelet shape change through regulation of the RhoA‐Rho Kinase‐MLC phosphatase signaling pathway

Background Platelet shape change, spreading and thrombus stability require activation of the actin cytoskeleton contractile machinery. The mechanisms controlling actin assembly to prevent unwanted platelet activation are unclear. Objectives We examined the effects of nitric oxide on the signaling pathways regulating platelet actin-myosin activation. Results S-nitrosoglutathione (GSNO) inhibited thrombin-induced platelet shape change and myosin phosphorylation of the myosin light chain (MLC). Because thrombin stimulates phospho-MLC through the RhoA/ ROCK dependent inhibition of MLC phosphatase (MLCP) we examined the effects of NO on this pathway. Thrombin caused the GTP loading and activation of RhoA, leading to the ROCK-mediated phosphorylation of MLCP on threonine 853 (thr853 ), which is known to inhibit phosphatase activity. Treatment of platelets with GSNO blocked ROCK-mediated increases in phosphoMLCP-thr853 induced by thrombin. This effect was mimicked by the direct activator of protein kinase G, 8-pCPT-PET-cGMP, and blocked by the inhibition of guanylyl cyclase, but not inhibitors of protein kinase A. Further exploration of the mechanism demonstrated that GSNO stimulated the association of RhoA with protein kinase G (PKG) and the inhibitory phosphorylation (serine188) of RhoA in a cGMP-dependent manner. Consistent with these observations, in vitro experiments revealed that recombinant PKG caused direct phosphorylation of RhoA. The inhibition of RhoA by GSNO prevented ROCK-mediated phosphorylation and inhibition of MLCP activity. Conclusions These data suggest novel crosstalk between the NO-cGMP-PKG and RhoA/ROCK signaling pathways to control platelet actin remodeling.

RhoA and Rac1 Gtpases Differentially Regulate Agonist-Receptor Mediated ROS Generation in Platelets

Agonist induced generation of reactive oxygen species (ROS) including superoxide anion (O-2) and hydrogen peroxide (H2O2) enhance platelet aggregation and hence the risk of thrombosis. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Previous studies have shown that small GTPases Rac1 and RhoA are involved in NOX activation. Rac GTPase activates NOX by directly binding to NOX as well as by interacting with p67phox to promote its binding to NOX (Physiol Rev 87: 245–313, 2007), whereas RhoA triggers ROS generation via the ROCK/p38MAPK cascade mediated phosphorylation of p47phox, a critical component of the NOX complex, (Exp Mol Med 37:575-87, 2005). To date, however, the roles of Rac1 and RhoA in platelet ROS production remain unclear. This study was conducted to define the contributions of Rac1- and RhoA- signaling to ROS generation and platelet function. ROS generation was quantified by flow cytometry in dcf-da (10 µM) loaded washed platelets. Thrombin has been shown to generate ROS in human platelets (Blood 106: 2757-2760, 2005). In this study we confirmed that platelets stimulated with thrombin generate ROS in a time- and a concentration- dependent manner. Addition of thrombin to human platelets pre-treated with NSC23766, a Rac-specific inhibitor, or murine platelets with Rac1 gene deletion, produced significantly less ROS than the matching control samples. Further, Phox-I, a pharmacologic inhibitor of Rac-p67phox interaction (Chem Biol 19: 228-24, 2012), potently suppressed thrombin induced ROS production, indicating that a Rac1-p67phox signaling axis is involved in thrombin mediated ROS production. Separately, treatment of washed human platelets with a RhoA specific inhibitor, Rhosin (Chem Biol 19:699-710, 2012) resulted in: (a) inhibition of the U46619, a stable analog of TXA2, induced activation of RhoA, but not that of Rac1or Cdc42; (b) U46619 induced phosphorylation p38MAPK and p47phox; and (c) U46619 or thrombin induced ROS generation. We further investigated the role of RhoA/ROCK/p38MAPK in ROS production by using platelets from RhoA-/- mice, Y27632 (a ROCK inhibitor) and SB203580 (a p38MAPK inhibitor). RhoA-/- platelets or human platelets treated with Y27632 or SB203580 exhibited significantly diminished ROS generation in response to thrombin. Next, we investigated the physiological effects of Rhosin on platelet activation. A pre-incubation of washed human platelets with Rhosin inhibited U46619 or thrombin induced platelet shape change, release of P-selectin, secretion of ATP and aggregation. The anti-platelet effects of Rhosin were reversible as washing of platelets after incubation with Rhosin abolished the inhibitory effect of Rhosin on platelet aggregation. These results suggest that (a) RhoA signaling, through ROCK/MAPK/p47phox activation, leads to ROS generation and platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC, and (b) Rac1 and RhoA differentially regulate platelet ROS generation by directly binding to NOX, promoting binding of p67phox to NOX and by phosphorylation of p47phox, respectively. DisclosuresNo relevant conflicts of interest to declare.

Dipyridamole synergizes with nitric oxide to prolong inhibition of thrombin-induced platelet shape change

We and others have previously demonstrated that nitric oxide (NO)-induced inhibition of platelet shape change is important in regulating platelet adhesion and aggregation, and therapeutic intervention of this pathway is clinically relevant for secondary prevention of stroke with dipyridamole. In the present study, we investigated whether dipyridamole affected the shape change of aspirinated platelets. Platelet shape change was inhibited using both authentic NO and sodium nitroprusside, as monitored by light scattering and mean platelet volume measurements. Dipyridamole synergized with NO, even at supra-therapeutic levels, to inhibit thrombin-induced shape change and further potentiated cAMP dependent protein kinase (PKA) mediated phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser157, even without altered levels of platelet cAMP. The effect of dipyridamole on NO-inhibited shape change depended on cGMP synthesis as evaluated by inhibition of soluble guanylyl cyclase. Measured increases in cGMP levels by dipyridamole and NO was assessed by mathematical modeling and found to be consistent with inhibition of phosphodiesterase 5 (PDE5). The model could explain the unexpected efficiency of dipyridamole in inhibiting PDE5 at the measured cGMP levels, by the majority of cGMP being bound to cGMP-dependent protein kinase (PKG). Still, selective activators of PKG failed to extend NO-mediated inhibition of the thrombin-induced platelet shape change, suggesting that PKG was not responsible for the inhibitory effect of NO and dipyridamole on shape change. The effects of dipyridamole were independent of the prostanoid and ADP pathways. Thus, the effect of dipyridamole on NO-mediated inhibition of platelet shape change may be an important and additional beneficial therapeutic effect of dipyridamole, which we suggest, is acting though localized amplification of the NO/cGMP/Phosphodiesterase3/cAMP/PKA-pathway. Probably, the efficiency of dipyridamole could be amplified clinically with NO donors.

Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide

AbstractThe thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change. (Blood. 2004;104:2775-2782)

A low molecular weight heparin, nadroparin (Fraxiparine), inhibits thrombin-induced platelet shape change and does not enhance spontaneous platelet aggregation.

Therapeutic concentrations, added in vitro, of a low molecular weight heparin (LMWH), nadroparin (Fraxiparine), inhibit thrombin-induced platelet shape change (PSC), an early stage of human platelet activation. PSC was monitored by measuring the median platelet volume (MPV) using a high resolution channelyzer. The inhibitory action of nadroparin was specific to thrombin since this LMWH did not influence PSC induced by other agonists. In whole blood, unfractionated heparin (UH) but not nadroparin, significantly enhanced spontaneous platelet aggregation and increased MPV when compared with LMWH or saline controls. In conclusion, the LMWH, nadroparin, exerts less platelet activation than UH.

A low molecular weight heparin, nadroparin (FRAXI-PARINE), inhibits thrombin-induced platelet shape change

Department of Chemical Pathology & Human Metabolism, Royal Free Hospital & School of Medicine (University of London), Pond Street, London NW3 2QG, UK

Structurally distinct disintegrins contortrostatin and multisquamatin differentially regulate platelet tyrosine phosphorylation.

Tyrosine phosphorylation of multiple platelet proteins is regulated by the integrin alpha IIb beta 3. In order to further examine integrin-regulated tyrosine phosphorylation, we have used small Arg-Gly-Asp-containing snake venom proteins (termed disintegrins) that inhibit platelet aggregation to competitively block the agonist-induced binding of fibrinogen to alpha IIb beta 3. One structurally unique disintegrin, contortrostatin (which appears to be a disulfide-linked dimer of 13.5 kDa with two Arg-Gly-Asp sites), was found to trigger signaling events typically mediated by fibrinogen cross-linking of alpha IIb beta 3, as demonstrated by tyrosine phosphorylation of the tyrosine kinase pp72syk and a 140-kDa protein. Contortrostatin and another disintegrin, multisquamatin (a monomer of 5.7 kDa with a single Arg-Gly-Asp site), did not affect thrombin-induced platelet shape change, secretion, or integrin-independent tyrosine phosphorylation; however, they inhibited aggregation and aggregation-dependent tyrosine phosphorylation of numerous proteins, including the focal adhesion kinase pp125FAK. Our results suggest that structurally distinct disintegrins have varying effects on tyrosine phosphorylation; while monomeric multisquamatin and dimeric contortrostatin both inhibit aggregation-dependent tyrosine phosphorylation, contortrostatin also possesses a unique functional activity that allows it to activate an intracellular signaling pathway leading to tyrosine phosphorylation. This activity may be involved in the function of this snake venom protein on hemostasis.

Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

Effects of exogenous phospholipids on platelet activation

Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and spiculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457–4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406–5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.

Immunologic Analysis of the Cloned Platelet Thrombin Receptor Activation Mechanism: Evidence Supporting Receptor Cleavage, Release of the N-Terminal Peptide, and Insertion of the Tethered Ligand Into a Protected Environment

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region ("tethered ligand") into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin-induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34-52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34-52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83-94). Anti-TR34-52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34-52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83-94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34-52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34-52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34-52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34-52 prevented cleavage of the TR34-52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

High Molecular Weight Kininogen Inhibits Thrombin-Induced Platelet Aggregation and Cleavage of Aggregin by Inhibiting Binding of Thrombin to Platelets

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

5′- p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of M r 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [ 3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [ 3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy- d-glucose, d-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca 2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (chelator of Ca 2+) inhibited platelet aggregation and cleavage of aggregin in [ 3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10–20 μ m, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca 2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [ 3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [ 3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.