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Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing αIIbβ3 integrin activation
Abstract
Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin αIIbβ3 through proteolysis of the αIIb subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P2) accumulation. PtdIns(3,4)P2 has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained αIIbβ3 integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of αIIbβ3 activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where αIIbβ3 integrin remains activated.
... Conversely, the possibility of small foci of tissue injury caused by the ligament trauma itself and the distension of the adjacent vascular wall, which could lead to the release of tissue factor and adhesion and platelet activation, should also be considered in this complex system 58,59 . In this situation, the inhibition of CG and HNE by rBbCI-His (6) may interfere with the contribution of platelets for the development of thrombi by inhibiting the cleavage of α2β3 integrin by HNE hindering the binding of fibrinogen to integrin 60,61 . Additionally, the inhibited CG prevents the cleavage of PAR4, and activation of local platelet aggregation is will be reduced 62 . ...
The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His(6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His(6), heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His(6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His(6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His(6) (1.40 mg/kg), with the rBbCI-His(6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His(6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His(6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.
Monozyten und Leukozyten interagieren mit dem Hämostasesystem über verschiedene Mechanismen. Aktivierte Monozyten und Leukozyten können das plasmatische Gerinnungssystem durch die Expression von Tissue Factor aktivieren. Die daraus resultierende Fibrinbildung trägt dazu bei, den Entzündungsprozess zu begrenzen, und behindert die Ausbreitung von Bakterien und anderen Krankheitserregern. Durch direkten zellulären Kontakt können Monozyten und Leukozyten Endothelzellen aktivieren und eine prokoagulatorische Antwort induzieren. Dies wird durch die Sezernierung von Zytokinen und anderen proinflammatorischen Mediatoren verstärkt, die neben der Endothelzellaktivierung die hepatische Synthese von Gerinnungsfaktoren stimulieren können. Weiterhin sind Monozyten und Leukozyten eine Hauptquelle für die Bildung von an Tissue Factor reichen Mikropartikeln. Diese ermöglichen die Aufrechterhaltung einer basalen Gerinnungsaktivierung, sind in die Gerinnselbildung involviert und spielen wahrscheinlich eine wichtige Rolle in der Pathogenese von thromboembolischen Prozessen.
Background
Pulmonary exposure to nanoparticles (NPs) may affect, in addition to pulmonary toxicity, the cardiovascular system such as procoagulant effects, vascular dysfunction and progression of atherosclerosis. However, only few studies have investigated hemostatic effects after pulmonary exposure.Methods
We used Bmal1 (brain and muscle ARNT-like protein-1) knockout (Bmal1¿/¿) mice which have a disturbed circadian rhythm and procoagulant phenotype, to study the pulmonary and hemostatic toxicity of multi-walled carbon nanotubes (MWCNTs) and zinc oxide (ZnO) NPs after subacute pulmonary exposure. Bmal1¿/¿ and wild-type (Bmal1+/+) mice were exposed via oropharyngeal aspiration, once a week, during 5 consecutive weeks, to a cumulative dose of 32 or 128 ¿g MWCNTs or 32 or 64 ¿g ZnO NPs.ResultsMWCNTs caused a pronounced inflammatory response in the lung with increased cell counts in the broncho-alveolar lavage and increased secretion of interleukin-1ß and cytokine-induced neutrophil chemo-attractant (KC), oxidative stress (increased ratio of oxidized versus reduced glutathione and decreased total glutathione) as well as anemic and procoagulant effects as evidenced by a decreased prothrombin time with increased fibrinogen concentrations and coagulation factor (F)VII. In contrast, the ZnO NPs seemed to suppress the inflammatory (decreased neutrophils in Bmal1¿/¿ mice) and oxidative response (increased total glutathione in Bmal1¿/¿ mice), but were also procoagulant with a significant increase of FVIII. The procoagulant effects, as well as the significant correlations between the pulmonary endpoints (inflammation and oxidative stress) and hemostasis parameters were more pronounced in Bmal1¿/¿ mice than in Bmal1+/+ mice.Conclusions
The Bmal1¿/¿ mouse is a sensitive animal model to study the procoagulant effects of engineered NPs. The MWCNTs and ZnO NPs showed different pulmonary toxicity but both NPs induced procoagulant effects, suggesting different mechanisms of affecting hemostasis. However, the correlation analysis suggests a causal association between the observed pulmonary and procoagulant effects.
We used proteomics to identify systematic changes in the plasma proteins of patients undergoing coronary artery bypass grafting (CABG) by means of cardiopulmonary bypass surgery. It is known that, after CABG, a complex systemic inflammatory responses ensues that favors the occurrence of adverse postoperative complications frequently recognizing inflammation itself and/or thrombosis as the underlying mechanism. We found a marked and persistent postoperative increase in the levels of the serpin-protease inhibitor alpha(1)-antichymotrypsin (alpha(1)-ACT) that fully maintains the inhibitory activity blunting its protease substrate cathepsin G. An intraoperative increase followed by a rapid decline in proteases activation was documented, accompanied by a substantial induction of leucine-rich-alpha-2-glycoprotein, a protein involved in neutrophilic granulocyte differentiation. Finally, a time-dependent alteration in the expression of haptoglobin, transthyretin, clusterin, and apoE was observed. In conclusion, we showed that after CABG, a protease/antiprotease imbalance occurs with early cathepsin G activation and a more delayed increase in alpha(1)-ACT. As cathepsin G is a serpin involved both in inflammation and coagulation activation, this confirms and expands the concept of a marked dysregulation of both inflammatory and hemostatic balances occurring after CABG. The pharmacologic modulation of this imbalance may be a new therapeutic target to reduce postoperative complications.
The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.
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Inflammation and thrombosis are related via interactions between leukocytes, platelets, the vasculature, and the coagulation system. However, the mechanisms behind these interactions remain poorly understood. Objectives: We have investigated the effects of the well known pulmonary inflammation induced by silica for the development of peripheral thrombogenicity in a hamster model of thrombosis. In addition, the consequences of pulmonary macrophage and circulating monocyte and neutrophil depletion on the thrombogenicity were investigated.
Silica particles (2-200 mug/hamster) were intratracheally instilled, and experimental thrombosis in photochemically induced femoral vein lesions was assessed 24 hours later, in association with cellular infiltration in the lung.
Intratracheally instilled silica particles (20 and 200 mug/hamster) triggered pulmonary inflammation, together with stimulation of peripheral platelet-rich thrombus formation. Both the selective depletion of lung macrophages by intratracheal administration of clodronate liposomes and the combined depletion of circulating monocytes and neutrophils by intraperitoneal injection of cyclophosphamide significantly reduced silica-induced influx of macrophages and neutrophils in bronchoalveolar lavage, and reduced peripheral thrombogenicity. Silica-induced lung inflammation was accompanied by increased neutrophil elastase levels in bronchoalveolar lavage and in plasma. Specific neutrophil elastase inhibition in the lung did not affect lung inflammation but reduced peripheral thrombogenicity.
These findings uncover pulmonary macrophage-neutrophil cross-talk releasing neutrophil elastase into the blood circulation. Elastase, triggering activation of circulating platelets, may then predispose platelets to initiate thrombotic events on mildly damaged vasculature.
Platelets contain and release some matrix metalloproteinases (MMPs), enzymes involved in the degradation of extracellular matrix, and one of these (MMP-2) exerts a proaggregatory effect. We explored the signal transduction mechanisms activated by MMP-2 in human blood platelets.
Recombinant, human MMP-2, added before stimulation with subthreshold doses of different agonists, potentiated platelet activation, calcium influx, IP3 formation, and pleckstrin phosphorylation. Wortmannin and LY29400, two PI3-K inhibitors, suppressed the potentiating effects of MMP-2 and preincubation with MMP-2 enhanced the thrombin-induced association of the p85alpha PI3-K subunit with the cytoskeleton and increased the phosphorylation of PKB. Protein tyrosine kinase inhibitors, MAP kinase inhibitors, PLA2 inhibitors, cyclooxygenase inhibitors and antagonists of the P2Y1 and P2Y12 receptors did not affect the potentiating activity of MMP-2 on platelets.
Our data show that MMP-2, at a concentration released by activated platelets, facilitates platelet activation acting at the level of a second messenger system common to different agonists and related to the activation of PI3-K. Platelet-released MMP-2 may contribute to platelet activation in vivo.
The consequences of arterial thrombosis such as myocardial infarction, stroke and peripheral vascular occlusion are the leading causes of morbidity and mortality. A high leukocyte count and an elevation in inflammatory markers are identified as significant risk factors for thrombosis. Leukocytes form the front line in defense against infection and are the first cells arriving at the site of inflammation. This review summarizes the cellular and molecular mechanisms by which adherent leukocytes can induce a prothrombotic state.
Circulating tissue factor has been recognized as a potential prothrombotic factor initiating thrombosis after vascular injury. The tissue factor is present on microvesicles originated from activated leukocytes. Leukocytes generate tissue factor containing microvesicles following stimulation with cytokines and following platelet adhesion via P-selectin. Additionally, activated leukocytes release several mediators, such as cathepsin G and elastase, which can activate both the coagulation cascade and platelets. Furthermore, new roles for leukocytes have been identified in vascular injury in sickle cell anemia, in vascular occlusion following the rupture of atherosclerotic plaque, and in thrombotic complications of myeloproliferative diseases.
Leukocyte adhesion to endothelium and platelets plays an important role in the activation of the coagulation cascade. An excessive activation of leukocytes during the inflammatory process may induce a systemic procoagulant state. Elucidation of critical steps in activation of coagulation by leukocytes may offer a new therapeutic target for antithrombotic therapy based on blocking leukocyte adhesion.
Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet-dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein-coupled receptors, the Gq-coupled P2Y1 receptor, and the Gi-coupled P2Y12 receptor. ATP, on the other hand, acts through the ligand-gated channel P2X1. Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A2 generation. ADP-induced dense granule release depends on generated thromboxane A2. Furthermore, costimulation of both P2Y1 and P2Y12 receptors is required for ADP-induced platelet aggregation. ATP stimulation of P2X1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.
Negative pressure therapy has been recently used for managing lymphatic or infective groin complications. The aim of this study was to investigate any possible association between application of negative pressure therapy in the groin area and deep-vein thrombosis. Acute surgical wounds were created at the inguinal areas in 7 pigs. Different negative pressures ranging from -50 to -200 mmHg were applied directly over the femoral vessels, and blood flow alterations were studied using a Doppler ultrasound. Femoral vein specimens were also removed for histological examination after 12 hours of therapy. It has been demonstrated that negative pressure therapy does not significantly alter the baseline lower limb venous return. Histology demonstrated several changes, which are associated with vein thrombogenesis. The hemodynamic and pathological findings still leave a potential for thrombogenic effects of negative pressure therapy and warrant care to protect the femoral veins, with the use of thrombosis prophylaxis measures.
In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of alpha(IIb)beta(3) and alpha(2)beta(1) regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y(12) receptors provokes only transient alpha(IIb)beta(3) activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, alpha(IIb)beta(3) can be secondarily inactivated in platelets with prolonged high Ca(2+) rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin alpha(2)beta(1) is found to be activated by a mechanism that is directly linked to alpha(IIb)beta(3) activation. Integrin alpha(2)beta(1) can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.
Of the four known protease-activated receptors (PARs), PARI and PARE are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists, Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These data show that PARI mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.
32P-Labeled human platelets were incubated with thrombin (1 unit/ml) for 5 min at 37 degrees C under conditions allowing maximal synthesis of [32P]phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2). Incorporation of 32P into the latter phosphoinositide was dose-dependently reduced (to a maximal level averaging 60%) by the tetrapeptide RGDS, an inhibitor of fibrinogen binding to activated glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin). Identical results were obtained with the fibrinogen gamma-chain dodecapeptide HHLGGAKQAGDV, whereas the tripeptide RGD and the tetrapeptide RGES displayed reduced or undetectable effects on 32P labeling of PtdIns(3,4)P2, respectively, in good correlation with their ability to inhibit platelet aggregation and fibrinogen binding to activated alpha IIb-beta 3 integrin. In addition, pathological platelets from three patients suffering thrombasthenia, which lack alpha IIb-beta 3 integrin and fail to aggregate in response to thrombin, displayed hardly detectable increases in the 32P labeling of PtdIns(3,4)P2. In contrast, thrombin-stimulated synthesis of PtdIns(3,4)P2 was unaltered in other deficient platelets lacking the glycoprotein Ib-IX complex (Bernard-Soulier syndrome). Although additional pathways seem to be involved in the regulation of phosphatidylinositol-3-kinase, these data indicate a strong relationship between platelet aggregation involving fibrinogen binding to activated alpha IIb-beta 3 integrin and the synthesis of the novel phosphoinositides phosphorylated at position D-3 of the inositol ring.
Platelets contain a pool of endogenous adhesive proteins that can be released and may bind to surface membrane receptors under appropriate conditions. Because the binding of exogenous fibrinogen to platelets was shown previously to be accompanied by a time-dependent decrease in fibrinogen accessibility to antibody and enzymes, studies were performed to evaluate changes in the expression of endogenous fibrinogen released from thrombin-stimulated platelets using monospecific polyclonal and monoclonal antibody F(ab')2 fragments. Parallel studies were performed to compare the expression of released fibronectin and von Willebrand factor (vWF). Binding of polyclonal antibody F(ab')2 fragments directed against individual adhesive proteins was inhibited by EDTA or the 10E5 monoclonal antibody, suggesting that fibrinogen, fibronectin, and vWF expression was mediated, in large part, by divalent cation-dependent interactions with the glycoprotein IIb-IIIa complex. Interestingly, when polyclonal antibody F(ab')2 fragments were added to platelet suspensions at discrete times after thrombin stimulation, antifibrinogen F(ab')2 binding decreased by 72% +/- 15% (mean +/- SD, n = 22) over a 60-minute time course, whereas antifibronectin and anti-vWF antibody F(ab')2 fragment binding changed minimally (6% +/- 23%, n = 22 and 3% +/- 26%, n = 14, respectively). Similar observations were made with monoclonal antibodies. Parallel experiments using 125I-labeled fibrinogen as a marker indicated that the observed decrease in antifibrinogen F(ab')2 binding was not accompanied by fibrinogen dissociation. Moreover, antibody accessibility to platelet-bound fibrinogen could be restored after Triton X-100 platelet lysis. The data suggest that fibrinogen, fibronectin, and vWF are not coordinately expressed on thrombin-stimulated platelets. Rather, fibrinogen expression appears transient compared with the expression of fibronectin and vWF. The ability of platelets to secrete and organize adhesive proteins on their surface is likely to have important implications for hemostasis and thrombosis.
Occupancy of integrin receptors induces conformational changes in the receptor, resulting in exposure of novel interactive sites termed ligand-induced binding sites (LIBS). We report here that Fab fragments of certain antibodies against LIBS on integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) block platelet aggregation. Thus, certain LIBS or the regions surrounding them may participate in events required for platelet aggregation. In addition, certain anti-alpha IIb beta 3 LIBS Fab fragments stimulated platelet aggregation. This was due to induction of fg binding to alpha IIb beta 3, apparently by shifting a conformational equilibrium between a "resting" and an "activated" state of alpha IIb beta 3. Some of the activating anti-LIBS Fab fragments also induced high affinity fibronectin binding to alpha IIb beta 3, whereas others did not. Thus, changes in the conformation of this integrin modulate both the specificity and affinity of ligand recognition.
Radioactive PtdIns(3)P was detected in human platelets incubated with [32P]Pi, but remained unaffected by thrombin treatment. In contrast, [32P]PtdIns(3,4)P2 was absent from resting platelets, but was produced by thrombin-activated platelets in a dose- and time-dependent manner. [32P]PtdInsP3 was never found under these conditions. These changes are similar to those elicited in other cells by platelet-derived growth factor or the oncogene product pp60c-src.
The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.
A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.
Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.
Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.
We have focused our interest on the platelet-activating properties of two polymorphonuclear neutrophil (PMN)-derived proteinases, namely elastase (HLE) and cathepsin G (Cat.G). First of all, we observed that whereas HLE was unable to trigger platelet activation by itself, it enhanced platelet activation induced by Cat.G when both proteinases were added simultaneously. It has been recently described that, upon stimulation, PMN released Cat.G, which in turn activated surrounding platelets. Thus, we looked for a combined effect of Cat.G and HLE during this cell-to-cell interaction. When PMN (5 x 10(6)/mL) were stimulated by 0.5 mumol/L N-formyl-Met-Leu-Phe, they released 237.9 +/- 49.1 nmol/L Cat.G and 381.7 +/- 28.0 nmol/L HLE. Such a concentration of purified Cat.G (240 nmol/L) induced only a moderate platelet activation when added to a PMN-platelet mixture. However, when Cat.G (240 nmol/L) and HLE (380 nmol/L) were added together, the resulting platelet activation was strictly comparable to that corresponding to the addition of N-formyl-Met-Leu-Phe (P > .05) in terms of aggregation, dense and alpha granule secretion, and thromboxane B2 production. In fact, Elafin, a specific HLE inhibitor, when added to the PMN-platelet cooperation system triggered by N-formyl-Met-Leu-Phe, prevented platelet activation within the same range of concentrations as for inhibition of HLE activity. In conclusion, we now show that not only Cat.G, but also HLE is involved in the PMN-mediated platelet activation.
Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.
The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.
Neutrophil elastase (NE) and cathepsin G are two serine proteinases released concomitantly by stimulated polymorphonuclear neutrophils. We previously demonstrated that while NE by itself does not activate human platelets, it strongly enhances the weak aggregation induced by a threshold concentration of cathepsin G (threshold of cathepsin G) (Renesto, P., and Chignard, M. (1993) Blood 82, 139-144). The aim of this study was to delineate the molecular mechanisms involved in this potentiation process. Two main pieces of data prompted us to focus on the activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. First, previous studies have shown this integrin to be particularly prone to proteolytic regulation of its function. Second, we found that the potentiating activity of NE on the threshold of cathepsin G-induced platelet aggregation was strictly dependent on the presence of exogenous fibrinogen. Using flow cytometry analysis, NE was shown to trigger a time-dependent binding of PAC-1 and AP-5, two monoclonal antibodies specific for the activated and ligand-occupied conformers of alphaIIbbeta3. Furthermore, the potentiated aggregation was shown to result from an increased capacity of platelets to bind fibrinogen. Indeed, the combination of NE and threshold of cathepsin G increased the binding of PAC-1 approximately 5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by approximately 3-fold in threshold of cathepsin G-stimulated platelets (p < 0.05). By contrast, phosphatidic acid accumulation, pleckstrin phosphorylation, and calcium mobilization produced by the combination of NE and threshold of cathepsin G were not significantly different from those measured with threshold of cathepsin G alone (p > 0.05), indicating that the phospholipase C/protein kinase C pathway is not involved in the potentiation of aggregation. The foregoing data, as well as the requirement of catalytically active NE to trigger alphaIIbbeta3 activation and potentiate threshold of cathepsin G-initiated platelet aggregation, led us to examine whether the structure of this integrin was affected by NE. Immunoblot and flow cytometry analysis revealed a limited proteolysis of the carboxyl terminus of the alphaIIb subunit heavy chain (alphaIIbH), as judged by the disappearance of the epitope for the monoclonal antibody PMI-1. Mass spectrometry studies performed on a synthetic peptide mapping over the cleavage domain of alphaIIbH predicted the site of proteolysis as located between Val837 and Asp838. Treatment by NE of ATP-depleted platelets or Chinese hamster ovary cells expressing human recombinant alphaIIbbeta3 clearly established that activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of alphaIIbH. In support of this latter observation, a close correlation was observed between the kinetics of proteolysis of alphaIIbH on platelets and that of expression of the ligand binding activity of alphaIIbbeta3 (r2 = 0.902, p </= 0. 005). However, only a subpopulation ( approximately 25%) of the proteolyzed alphaIIbbeta3 appeared to fully express the ligand binding capacity. Altogether, these results demonstrate that NE up-regulates the fibrinogen binding activity of alphaIIbbeta3 through a restricted proteolysis of the alphaIIb subunit, and that this process is relevant for the potentiation of platelet aggregation.
The aggregation of human platelets is an important physiological hemostatic event contingent upon receptor-dependent activation of the surface integrin alphaIIbbeta3 and subsequent binding of fibrinogen. Aggregating platelets form phosphatidylinositol 3, 4-bisphosphate (PtdIns(3,4)P2), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). It has been assumed that PtdIns(3,4)P2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P2 is formed after directly activating alphaIIbbeta3 with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P3, but to transient formation of PtdIns3P and generation of PtdIns(3,4)P2, the latter primarily by PtdIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P3. Integrin-activated generation of the second messenger PtdIns(3,4)P2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.
We have observed that aggregation of human platelets, caused by activation of integrin αIIbβ3 and its consequent binding of fibrinogen, stimulates a novel pathway for synthesis of phosphatidylinositol 3,4bisphosphate,
thereby activating protein kinase B/Akt. Such synthesis depends upon both the generation of phosphatidylinositol 3-phosphate
(PtdIns3P), which is sensitive to wortmannin (IC50 7 nm) and calpain inhibitors, and the phosphorylation of PtdIns3P by PtdIns3P 4-kinase. We now report that a recently characterized
C2 domain-containing phosphoinositide 3-kinase isoform (HsC2-PI3K) is present in platelets and a leukemic cell line (CHRF-288)
derived from megakaryoblasts, and is likely to be responsible for the stimulated synthesis of PtdIns3P observed in platelets.
HsC2-PI3K, identifiable by Western blotting and immunoprecipitatable activity, is sensitive to wortmannin (IC50 6–10 nm), requires Mg2+, and shows strong preference for PtdIns over PtdIns4P or phosphatidylinositol 4,5-bisphosphate as substrate. HsC2-PI3K is
activated severalfold when platelets aggregate in an αIIbβ3-dependent manner or when platelet or CHRF-288 lysates are incubated with Ca2+. Activation is prevented by calpain inhibitors. CHRF-288, which cannot undergo activation of αIIbβ3 and thereby aggregate in response to platelet agonists, do not generate PtdIns3P or activate HsC2-PI3K under conditions that
stimulate other phosphoinositide 3-kinases. HsC2-PI3K may thus be an important effector for integrin-dependent signaling.
Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.
Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents.
Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P2accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.
Platelets contain a pool of endogenous adhesive proteins that can be released and may bind to surface membrane receptors under appropriate conditions. Because the binding of exogenous fibrinogen to platelets was shown previously to be accompanied by a time-dependent decrease in fibrinogen accessibility to antibody and enzymes, studies were performed to evaluate changes in the expression of endogenous fibrinogen released from thrombin-stimulated platelets using monospecific polyclonal and monoclonal antibody F(ab')2 fragments. Parallel studies were performed to compare the expression of released fibronectin and von Willebrand factor (vWF). Binding of polyclonal antibody F(ab')2 fragments directed against individual adhesive proteins was inhibited by EDTA or the 10E5 monoclonal antibody, suggesting that fibrinogen, fibronectin, and vWF expression was mediated, in large part, by divalent cation-dependent interactions with the glycoprotein IIb-IIIa complex. Interestingly, when polyclonal antibody F(ab')2 fragments were added to platelet suspensions at discrete times after thrombin stimulation, antifibrinogen F(ab')2 binding decreased by 72% +/- 15% (mean +/- SD, n = 22) over a 60-minute time course, whereas antifibronectin and anti-vWF antibody F(ab')2 fragment binding changed minimally (6% +/- 23%, n = 22 and 3% +/- 26%, n = 14, respectively). Similar observations were made with monoclonal antibodies. Parallel experiments using 125I-labeled fibrinogen as a marker indicated that the observed decrease in antifibrinogen F(ab')2 binding was not accompanied by fibrinogen dissociation. Moreover, antibody accessibility to platelet-bound fibrinogen could be restored after Triton X-100 platelet lysis. The data suggest that fibrinogen, fibronectin, and vWF are not coordinately expressed on thrombin- stimulated platelets. Rather, fibrinogen expression appears transient compared with the expression of fibronectin and vWF. The ability of platelets to secrete and organize adhesive proteins on their surface is likely to have important implications for hemostasis and thrombosis.
Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.
Fibrinogen is required for ADP-induced aggregation of washed human platelets and enhances ADP-induced aggregation of rabbit platelets. To achieve these effects, fibrinogen had to be either present before ADP was added or added with the ADP. When fibrinogen was added after platelets had changed in shape in response to ADP, fibrinogen did not support ADP-induced aggregation. The association of 125I-fibrinogen with platelets during ADP-induced aggregation was investigated. A mixture of 125I-fibrinogen and trivalent 51Cr (as a measure of trapped fluid) was added to suspensions of washed human or rabbit platelets. Samples were taken for rapid centrifugation before and at several times after the addition of ADP. The percentage of labeled fibrinogen associated with the platelets was calculated. Fibrinogen associated transiently with platelets during their response to ADP. The largest amount of fibrinogen became associated with the platelets almost immediately after the addition of ADP; less fibrinogen was associated with the platelets at the peak of aggregation, and the associated fibrinogen was lost during platelet deaggregation. Association occurred only if conditions permitted platelet aggregation in response to ADP; inhibitory conditions included lack of divalent cations, inhibition of platelet metabolism, or the presence of inhibitors of platelet aggregation such as prostaglandin E 1 or adenosine. Thus the association of fibrinogen with platelets appeared to take place during the initial changes induced by ADP. These results indicate that fibrinogen may be involved in transient linkages between platelets in ADP-induced aggregates.
We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.
Eglin C is an inhibitor of two serine proteinase-derived polymorphonuclear leucocytes (PMN) i.e., elastase and cathepsin G. Since the latter has recently been shown to be involved in the activation of platelets by stimulated PMN, the effects of recombinant eglin C in the PMN-platelet cooperation model were studied. First, the inhibitory capacity of eglin C against purified cathepsin G was measured spectrophotometrically by following hydrolysis of a specific synthetic substrate, N-succinyl-ala-ala-pro-phe-para-nitroanilide. The inhibition of the enzymatic activity of 180 nM (5 micrograms/ml) cathepsin G was directly proportional to eglin C concentration and reached 100% with 2 micrograms/ml (240 nM). Platelet activation generated by a submaximal concentration of cathepsin G (200 nM) was also totally suppressed by 2 micrograms/ml of eglin C. Inhibition was specific (a 100 times higher concentration of eglin C did not alter platelet activation induced by thrombin), and surmountable (an increase of cathepsin G concentration reduced the eglin C effect). Thus, the mechanism of inhibition by eglin C of cathepsin G-induced platelet activation could be explained by a stoichiometric relation between eglin C and cathepsin G as previously described. Investigations were then performed with the PMN-platelet cooperation model, using two distinct stimuli, N-formylmethionylleucylphenylalanine (FMLP) or recombinant human C5a, at submaximal concentrations, 2.10(-7) M and 10(-7) M, respectively. A concentration-dependent inhibition of platelet activation aggregation and serotonin release-lambda was observed. Eglin C used at 10 micrograms/ml and 25 micrograms/ml totally blocked the platelet responses induced by recombinant human C5a and FMLP, respectively. Leucotriene B4, but also thromboxane B2 production measured by radioimmunoassays, were observed under FMLP activation. In the presence of eglin C, thromboxane B2 formation was totally suppressed, whereas leucotriene B4 synthesis was still effective. In fact, the mechanism of inhibition of eglin C is located neither on PMN (leucotriene B4 formation by FMLP-activated PMN was not affected), nor on platelets (response to thrombin was unchanged). The target is most probably cathepsin G since eglin C suppressed thromboxane B2 formation by platelets challenged by this serine proteinase. These results constitute an argument in favor of the implication of cathepsin G in the PMN-mediated platelet activation. Moreover, they reinforce the hypothesis that this mechanism could be operating under in vivo pathologic conditions, since eglin C is capable of preventing or ameliorating some experimental pulmonary diseases.
Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.
Although proteases are traditionally viewed as degradative enzymes, characterization of a family of G protein-coupled receptors that are activated by proteolysis reveals a new role. Certain proteases function as signaling molecules that specifically regulate cells by cleaving and activating a family of proteinase-activated receptors.
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