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 Mr 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 Ca2+ 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(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. 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.     

Aggregin: a platelet ADP receptor that mediates activation

ADP is known to induce platelet shape change, aggregation, and exposure of fibrinogen binding sites as well as inhibit stimulated adenylate cyclase. The platelet is unique in that its purinergic receptor prefers ADP over ATP, which functions as a competitive antagonist. The affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), has been used to covalently label a single membrane protein, aggregin, on the external platelet surface with mol wt of 100 kDa. Concomitant with incorporation of FSBA, ADP-induced shape change, aggregation, and fibrinogen binding is inhibited. FSBA is also a weak agonist at short times and high concentration, which suggests that prior noncovalent binding to aggregin takes place before covalent modification. Aggregin differs from platelet glycoprotein IIIa in its physical and immunochemical properties. Aggregin is distinct from the receptor coupled to adenylate cyclase. Using FSBA as a probe, platelet aggregation by thromboxane A2 analogs and collagen was shown to be dependent on ADP but not the shape change induced by these agonists. Binding to aggregin is required for epinephrine-induced aggregation. In turn, epinephrine increases the affinity of ADP for its receptor. Thrombin at concentrations greater than 2 nM (0.2 units/ml) stimulates platelet aggregation independent of ADP, but by raising cytoplasmic Ca2+ it activates platelet calpain, which in turn cleaves aggregin. Thus aggregin, in addition to serving as the ADP receptor linked to shape change and aggregation, plays a role in fibrinogen receptor latency that is relieved entirely by ADP binding to or proteolysis of aggregin.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Inhibition of ADP-induced platelet activation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole: Covalent modification of aggregin,a putative ADP receptor

ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-Cl. NBD-Cl inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-Cl also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-Cl did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-Cl blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I₂). NBD-Cl was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [¹⁴C]-NBD-Cl showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [¹⁴C]-NBD-Cl. These results (1) indicate that covalent modification of aggregin by NBD-Cl contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin. © 1996 Wiley-Liss, Inc.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

Inhibition of ADP-induced platelet shape change and aggregation by o-phthalaldehyde: evidence for covalent modification of cysteine and lysine residues

Platelets play a major role in the hemostatic process following vascular injury. Chemical modification of cysteine and/or lysine residues in platelet proteins has been shown to cause loss of platelet aggregation induced by diverse agonists; however, these investigations have not addressed the identity of the specific proteins affected. o-Phthalaldehyde (OPTH) is a unique chemical modification reagent that forms and permits the identification of fluorescent isoindole derivatives with proteins by covalently and simultaneously modifying closely spaced cysteine and lysine residues. We found that OPTH inhibited platelet aggregation induced by ADP, collagen, and U46619 (an analog of prostaglandin H2), but had minimal effect on platelet aggregation induced by thrombin, plasmin, chymotrypsin, A23187 (a calcium ionophore), PMA (phorbol 12-myristate 13-acetate), and PMA + A23187. Since platelet aggregation induced by ADP, collagen, and U46619 has been shown to involve binding of endogenous or exogenous ADP to the platelet receptor, our further studies focused on platelet aggregation induced by ADP. OPTH inhibited ADP-induced shape change and aggregation in a concentration-dependent manner. The second-order rate constant for the inhibition of ADP-induced platelet shape change (Ksc = 1.0 X 10(3) M-1 s-1) was lower than that for aggregation (Kagg = 5.4 X 10(3) M-1 s-1). Fluorescence excitation and emission spectra of OPTH-platelet adduct exhibited maxima at 346 and 437 nm, respectively, consistent with the formation of an isoindole derivative(s). The nonpenetrating thiol-specific reagent, p-chloromercuribenzenesulfonate (pCMBS) (0.8 mM), is known to block the inhibition of stimulated adenylate cyclase induced by ADP but not the ADP-induced platelet shape change. The inhibition of ADP-induced platelet shape change (Ksc = 1.5 X 10(3) M-1 s-1) by OPTH was not affected by pCMBS. OPTH, at concentrations (15-50 microM) that inhibited ADP-induced platelet aggregation and shape change did not raise the intracellular levels of adenosine cyclic 3',5'-monophosphate (cAMP) in platelets nor did it impair the ability of iloprost (a stable analog of prostaglandin I2) to raise the platelet cAMP level. Thus, OPTH under these conditions did not interact with platelet adenylate cyclase. 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been previously shown to inhibit ADP-induced platelet shape change and aggregation by covalently modifying aggregin (Mr = 100 kDa), a putative ADP receptor on platelet surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding

The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.     

Inhibition of epinephrine-induced platelet aggregation by a derivative of wheat germ agglutinin

A nonagglutinating derivative of wheat germ agglutinin has been prepared and used as a probe to explore the initial events in platelet activation. The lectin derivative had no effect on platelet aggregation by adenosine diphosphate, collagen, ristocetin, wheat germ agglutinin or trypsin but aggregation induced by epinephrine or thrombin was inhibited. Unlike thrombin, the inhibition of aggregation by the derivative could not be overcome by increasing the concentration of epinephrine. The derivative did not affect the binding of [³H]dihydroergocryptine to platelets. A 74,000 dalton protein isolated from platelet membranes by lectin affinity chromatography strongly inhibited platelet activation by thrombin but not by epinephrine. The receptors for thrombin and for epinephrine on platelets are different but they are closely linked.     

Inhibition of collagen-induced platelet reactivity by DGEA peptide

Direct interactions between collagen, the most thrombogenic component of the extracellular matrix, and platelet surface membrane receptors mediate platelet adhesion and induce platelet activation and aggregation. In this process two glycoproteins are crucial: integrin alpha2beta1, an adhesive receptor, and GPVI, which is especially responsible for signal transduction. Specific antagonists of the collagen receptors are useful tools for investigating the complexity of platelet-collagen interactions. In this work we assessed the usefulness of DGEA peptide (Asp-Gly-Glu-Ala), the shortest collagen type I-derived motif recognised by the collagen-binding integrin alpha2beta1, as a potential antagonist of collagen receptors. We examined platelet function using several methods including platelet adhesion under static conditions, platelet function analyser PFA-100TM, whole blood electric impedance aggregometry (WBEA) and flow cytometry. We found that DGEA significantly inhibited adhesion, aggregation and release reaction of collagen activated blood platelets. The inhibitory effect of DGEA on static platelet adhesion reached sub-maximal values at millimolar inhibitor concentrations, whereas the specific blocker of alpha2beta1 - monoclonal antibodies Gi9, when used at saturating concentrations, had only a moderate inhibitory effect on platelet adhesion. Considering that 25-30% of total collagen binding to alpha2beta1 is specific, we conclude that DGEA is a strong antagonist interfering with a variety of collagen-platelet interactions, and it can be recognised not only by the primary platelet adhesion receptor alpha2beta1 but also by other collagen receptors.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.     

Adenosine diphosphate receptors on blood platelets: potential new targets for antiplatelet therapy

Platelets play a key role not only in physiological haemostasis, but also under pathological conditions such as thrombosis. Platelet activation may be initiated by a variety of agonists including thrombin, collagen, thromboxane or adenosine diphosphate (ADP). Although ADP is regarded as a weak agonist of blood platelets, it remains an important mediator of platelet activation evoked by other agonists, which induce massive ADP release from dense granules, where it occurs in molar concentrations. Thus, ADP action underlies a positive feedback that facilitates further platelet aggregation and leads to platelet plug formation. Additionally, ADP acts synergistically to other, even weak, agonists such as serotonin, adrenaline or chemokines. Blood platelets express two types of P2Y ADP receptors: P2Y(1) and P2Y(12). ADP-dependent platelet aggregation is initiated by the P2Y1 receptor, whereas P2Y(12) receptor augments the activating signal and promotes platelet release reaction. Stimulation of P2Y(12) is also essential for ADP-mediated complete activation of GPIIb-IIIa and GPIa-IIa, and further stabilization of platelet aggregates. The crucial role in blood platelet biology makes P2(Y12) an ideal candidate for pharmacological approaches for anti-platelet therapy.     

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor.     

A novel anti-platelet monoclonal antibody (3C7) specific for the complex of integrin alpha IIb beta3 inhibits platelet aggregation and adhesion

Activation or ligand binding induces conformational changes in alpha IIb beta3, resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated alpha IIb beta3 mutant (CHO alpha IIb beta3Delta717) as the immunogen. This IgG 2b kappa named 3C7 was specific for the complex of alpha IIb beta3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an anti-ligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another alpha IIb beta3 mutant (beta3Y178A) suggesting that the Cys177-Cys184 loop of beta3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC50s of 5.6 and 0.05 microg/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin alpha IIb beta3 and a potential anti-thrombotic agent.     

Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Resistance to thromboembolism in PI3Kgamma-deficient mice.

Platelet aggregation and subsequent thrombosis are the major cause of ischemic diseases such as heart attack and stroke. ADP, acting via G protein-coupled receptors (GPCRs), is an important signal in thrombus formation and involves activation of phosphoinositide 3-kinases (PI3K). When platelets from mice lacking the G protein-activated PI3Kgamma isoform were stimulated with ADP, aggregation was impaired. Collagen or thrombin, however, evoked a normal response. ADP stimulation of PI3Kgamma-deficient platelets resulted in decreased PKB/Akt phosphorylation and alpha(IIb)beta(3) fibrinogen receptor activation. These effects did not influence bleeding time but protected PI3Kgamma-null mice from death caused by ADP-induced platelet-dependent thromboembolic vascular occlusion. This result demonstrates an unsuspected, well-defined role for PI3Kgamma downstream of ADP and suggests that pharmacological targeting of PI3Kgamma has a potential use as antithrombotic therapy.     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

Savignygrin, a platelet aggregation inhibitor from the soft tick Ornithodoros savignyi, presents the RGD integrin recognition motif on the Kunitz-BPTI fold

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of alpha-CD41 (P2) to platelets, the binding of purified alpha(IIb)beta3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin alpha(v)beta3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward alpha(IIb)beta3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.     

Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain

Platelet glycoprotein (GP) VI has been proposed as the major collagen receptor for activation of human platelets. Human GPVI belongs to the immunoglobulin superfamily and is noncovalently associated with the FcRgamma chain that is involved in signaling through the receptor. In mice, similar mechanisms seem to exist as platelets from FcRgamma chain-deficient mice do not aggregate in response to collagen. However, the activating collagen receptor on mouse platelets has not been definitively identified. In the current study we examined the function and in vivo expression of GPVI in control and FcRgamma chain-deficient mice with the first monoclonal antibody against GPVI (JAQ1). On wild type platelets, JAQ1 inhibited platelet aggregation induced by collagen but not PMA or thrombin. Cross-linking of bound JAQ1, on the other hand, induced aggregation of wild type but not FcRgamma chain-deficient platelets. JAQ1 stained platelets and megakaryocytes from wild type but not FcRgamma chain-deficient mice. Furthermore, JAQ1 recognized GPVI (approximately 60 kDa) in immunoprecipitation and Western blot experiments with wild type but not FcRgamma chain-deficient platelets. These results strongly suggest that GPVI is the collagen receptor responsible for platelet activation in mice and demonstrate that the association with the FcRgamma chain is critical for its expression and function.