ADP-induced platelet aggregation and inhibition of adenylyl cyclase activity stimulated by prostaglandins: signal transduction mechanisms.

ADP is the oldest and one of the most important agonists of platelet activation. ADP induces platelet shape change, exposure of fibrinogen binding sites, aggregation, and influx and intracellular mobilization of Ca2+. ADP-induced platelet aggregation is important for maintaining normal hemostasis, but aberrant platelet aggregation manifests itself pathophysiologically in myocardial ischemia, stroke, and atherosclerosis. Another important aspect of ADP-induced platelet activation is the ability of ADP to antagonize adenylyl cyclase activated by prostaglandins. ADP-induced inhibition of the stimulated adenylyl cyclase activity does not appear to play a role in ADP-induced platelet aggregation in vitro or in vivo. It is believed that a single ADP receptor mediates the above two ADP-induced platelet responses in platelets. The ADP receptor mediating ADP-induced platelet aggregation and inhibition of the stimulated adenylyl cyclase activity has not been purified. Therefore, the nature of molecular mechanisms underlying the two seemingly unrelated ADP-induced platelet responses remains either unclear or less well understood. The purpose of this commentary is to examine and make suggestions concerning the role of phospholipases and G-proteins in the molecular mechanisms of signal transduction underlying the two ADP-induced platelet responses. It is hoped that such discussion would stimulate thinking and invite future debates on this subject, and energize investigators in their efforts to advance our knowledge of the details of the molecular mechanisms of ADP-induced platelet activation.

Phospholipase A2: its role in ADP- and thrombin-induced platelet activation mechanisms.

ADP and thrombin are two of the most important agonists of platelet aggregation--a cellular response that is critical for maintaining normal hemostasis. However, aberrant platelet aggregation induced by these agonists plays a central role in the pathogenesis of cardiovascular and cerebrovascular diseases. Agonist-induced primary or secondary activation of phospholipases leads to generation of the second messengers that participate in biochemical reactions essential to a number of platelet responses elicited by ADP and thrombin. Phospholipase A2 (PLA2) has been linked to cardiovascular diseases. However, the mechanism(s) of activation of PLA2 in platelets stimulated by ADP and thrombin has remained less well defined and much less appreciated. The purpose of this review is to examine and compare the molecular mechanisms of activation of PLA2 in platelets stimulated by ADP and thrombin.

Purinergic receptors in human blood platelets: chemical modification and cloning investigations.

Platelet aggregation is important for maintaining normal hemostasis. However, aberrant platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is one of the earliest and most important platelet agonists. ADP induces platelet aggregation, shape change, secretion, influx and intracellular mobilization of Ca2+, and inhibition of the adenylyl cyclase stimulated by prostaglandins. Binding of ADP to purinergic receptor(s) is required for elicitation of the ADP-induced platelet responses. But the platelet ADP receptor(s) has not been purified, largely due to the unavailability of the reagents that can be used to selectively label the platelet ADP receptor. The ADP receptor responsible for the ADP-induced platelet aggregation and inhibition of stimulated adenylyl cyclase activity has not been cloned due to difficulties in screening responsive clones generated from a cDNA library. Since the purified ADP-receptor protein is not available, antibodies that can be used as alternative tools to purify the ADP receptor or screen the clones expressing the receptor could not be made. In addition, the problem may be compounded by the low copy number and the susceptibility of the receptor to proteolysis. Therefore, signal transduction mechanisms underlying biochemical transformations in ADP-induced platelet responses remain less well defined and/less well understood. In the past decade efforts have been made to identify a platelet ADP receptor(s) by photoaffinity as well as affinity labeling by the ADP-affinity analogs. More recently efforts have been directed to clone the platelet ADP receptors. These investigations, however, have not produced definite results. The purpose of this review is to examine the results obtained by the photoaffinity- and affinity-labeling investigations and cloning experiments to identify a platelet ADP receptor(s).

Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets.

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses.

Modulation of platelet responses by 2-[3-(bromo-2-oxopropylthio)]adenosine-5'-diphosphate involves its binding to as well as covalent modification of an ADP-receptor, aggregin.

The 2-substituted ADP derivatives are known to activate human blood platelets with varying degrees of potency. For example, 2-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate [2-BDB-TADP], an ADP-affinity analog, was previously shown by us to be 50% as potent as ADP in inducing human blood platelet responses [Puri, R. N., Colman, R. F., and Colman, R. W. (1996) Eur. J. Biochem. 236, 862-870]. 2-Methylthio-ADP (2-MeS-ADP) has been known to be a far more potent agonist than ADP. However, the molecular basis for defining the rank order of potency of the 2-substituted ADP derivatives as agonists of platelet responses have been incompletely understood. We now report that 2-BOP-TADP (a one carbon atom lower homolog of 2-BDB-TADP) at equimolar concentration is as potent as ADP in inducing platelet responses. Prolonged incubation of platelets with 2-BOP-TADP abolished its ability to elicit cellular responses. An autoradiogram of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets labeled by incubating the platelets with 2-BOP-TADP for 1 h followed by reduction by NaB[3H]4 showed the presence of a single covalently radiolabeled protein band at 100 kDa. Preincubation of platelets with either ADP or ATP reduced the intensity of the band corresponding to the 100-kDa protein radiolabeled by 2-BOP-TADP and NaB[3H]4. The results show that (i) 2-BOP-TADP modulates ADP-induced platelet responses by interacting with aggregin and (ii) 2-BOP-TADP was twice as potent as 2-BDB-TADP, and (iii) the chain length of the substituent in a homologous series has an important bearing on the potency of a 2-substituted ADP analog.

ADP-induced platelet activation.

Platelet activation is central to the pathogenesis of hemostasis and arterial thrombosis. Platelet aggregation plays a major role in acute coronary artery diseases, myocardial infarction, unstable angina, and stroke. ADP is the first known and an important agonist for platelet aggregation. ADP not only causes primary aggregation of platelets but is also responsible for the secondary aggregation induced by ADP and other agonists. ADP also induces platelet shape change, secretion from storage granules, influx and intracellular mobilization of Ca2+, and inhibition of stimulated adenylyl cyclase activity. The ADP-receptor protein mediating ADP-induced platelet responses has neither been purified nor cloned. Therefore, signal transduction mechanisms underlying ADP-induced platelet responses either remain uncertain or less well understood. Recent contributions from chemists, biochemists, cell biologists, pharmacologists, molecular biologists, and clinical investigators have added considerably to and enhanced our knowledge of ADP-induced platelet responses. Although considerable efforts have been directed toward identifying and cloning the ADP-receptor, these have not been completely successful or without controversy. Considerable progress has been made toward understanding the mechanisms of ADP-induced platelet responses but disagreements persist. New drugs that do not mimic ADP have been found to inhibit fairly selectively ADP-induced platelet activation ex vivo. Drugs that mimic ADP and selectively act at the platelet ADP-receptor have been designed, synthesized, and evaluated for their therapeutic efficacy to block selectively ADP-induced platelet responses. This review examines in detail the developments that have taken place to identify the ADP-receptor protein and to better understand mechanisms underlying ADP-induced platelet responses to develop strategies for designing innovative drugs that block ADP-induced platelet responses by acting selectively at the ADP-receptor and/or by selectively interfering with components of ADP-induced platelet activation mechanisms.

A novel method for chemical modification of functional groups other than a carboxyl group in proteins by N-ethyl-5-phenylisooxazolium-3'-sulfonate (Woodward's reagent-K): inhibition of ADP-induced platelet responses involves covalent modification of aggregin, an ADP receptor.

The chemical reaction of N-ethyl-5-phenylisooxazolium-3'-sulfonate (Woodward's Reagent-K, WR-K) with a carboxyl group yields an enol ester that cannot be reduced by sodium borohydride in an aqueous solution, while other nucleophiles such as sulfhydryl, hydroxyl, amino, and imidazole groups, react with WR-K to yield unsaturated ketones that are capable of being reduced by sodium borohydride in an aqueous medium. Aggregin, a 100-kDa protein on the surface of human blood platelets has been identified as an ADP receptor. Autoradiography of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the samples of solubilized human blood platelets modified by WR-K and then reduced by tritiated sodium borohydride (NaB[3H]4) showed the presence of a prominent band corresponding to a 100-kDa radiolabeled protein. Labeling of platelets by WR-K and NaB[3H]4 was inhibited by ADP, ATP, and thiol group modifying reagents. WR-K blocked completely labeling of platelets by [beta-32P]-8-(4-bromo-2, 3-dioxo-butylthio)adenosine-5'-diphosphate, an ADP-affinity analog that selectively and covalently labels aggregin (Puri, R. N., Kumar, A., Chen, H., Colman, R. F., and Colman, R. W. (1995) J. Biol. Chem. 256, 24482-24488). WR-K also inhibited ADP-induced platelet shape change, aggregation, and mobilization of intracellular Ca2+ and blocked ADP-induced inhibition of stimulated adenylate cyclase activity. The results show conclusively that WR-K inhibited ADP-induced platelet responses by preventing binding of ADP to aggregin and suggest that ADP binding domain of aggregin contains an essential thiol group. The method of labeling proteins by WR-K and NaB[3H]4, hitherto not used to distinguish among functional groups modified by WR-K, offers a useful and convenient alternative to previously used ultraviolet spectral methods which cannot be used to investigate the modified proteins in intact cellular systems.

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-CI) 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-CI. NBD-CI inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-CI also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-CI did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-CI blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I2). NBD-CI 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-CI 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-CI. These results (1) indicate that covalent modification of aggregin by NBD-CI 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.

Platelet activation by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate is mediated by its binding to a putative ADP receptor, aggregin.

Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.

Inhibition of ADP-induced platelet responses by covalent modification of aggregin, a putative ADP receptor, by 8-(4-bromo-2,3-dioxobutylthio)ADP.

ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.

Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations.

Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Design and synthesis of a kininogen-based selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy or angioplasty for treatment of myocardial infarction. We previously demonstrated that thrombin-induced platelet aggregation is indirectly mediated by intracellularly activated calpain expressed on the platelet surface through the cleavage of aggregin, a putative ADP-receptor, and that high molecular weight kininogen (HK), a naturally occurring thiol protease inhibitor, modulates thrombin-induced platelet aggregation. Considering the substrate specificity of calpain and the conserved sequence in HK, we studied selective inhibitors of thrombin-induced platelet aggregation by the affinity labeling approach with an S-3-nitro-2-pyridinesulfenyl (Npys) group. H-Phe-Gln-Val-Val-Cys (Npys)-Gly-NH2, which combines chemical and structural features of calpain substrate specificity and the conserved sequence in HK, selectively inhibited thrombin-induced platelet aggregation. It did not inhibit the aggregatory effects of other platelet agonists, and did not inhibit amidolytic activity of thrombin and thrombin-induced platelet shape change. The design and synthesis of such inhibitors could lead to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation.

Specificity of the sequence in Phe-Gln-Val-Val-Cys (-3-nitro-2-pyridinesulfenyl)-Gly-NH2--a selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation is mediated in part by the intracellularly activated calpain expressed onto the external side of the membrane. We have previously shown that P1, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 [Npys = 3-nitro-2-pyridinesulfenyl], an affinity analog corresponding to the highly conserved sequence Gln-Val-Val-Ala-Gly-NH2, present in domains 2 and 3 of human kininogens, was an irreversible inhibitor of platelet calpain (second-order rate constant = 5.85 mM-1 s-1). P1 also selectively blocked thrombin-induced platelet aggregation. We have now synthesized twenty-three other peptides, analogous to P1, and evaluated them to define the specificity of the amino acid sequence in P1 to selectively block thrombin-induced platelet aggregation. We find that replacement by Leu of Val and by Tyr of Phe adjacent to Gln is minimally tolerated and the resulting peptides are partially effective in selectively blocking thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. Extensions of the N-terminal sequence in P1 did not improve its selectivity. Ac-Ala-Gln-Val-Val-Ala-Gly-NH2 (Ac, acetyl), a peptide containing the conserved sequence but lacking the Npys function, neither inhibited platelet calpain nor platelet aggregation induced by thrombin. Presence of the peptide sequence and Npys function are both required in P1 for its selective action in inhibiting platelet aggregation induced by thrombin.

Inactivation of yeast hexokinase by Cibacron brilliant red 3B-A.

Procion or Cibacron blue dyes, containing polynuclear aromatic rings and mono- and dichlorotriazine nuclei, immobilized on dextran matrices, have been used for over a decade to purify diverse groups of enzymes by dye-ligand chromatography. Comparatively less attention has been paid to investigating the nature of molecular interactions between similarly constituted red dyes and various enzymes so as to ascertain their potential and thus justify their use in the purification of enzymes by dye-ligand chromatography. We investigated and found that Cibacron brilliant red 3B-A, a monochlorotriazine dye, inhibited phosphotransferase activity of yeast hexokinase. The dissociation constant, KD, and the rate of dye-enzyme complex formation, k3, were 120 microM and 0.1 min-1, respectively. The enzyme was protected from inactivation by sugar and nucleotide substrates. About 2 mol of the dye bound per mole of the enzyme. The chromophore of the dye showed absorption at 524 nm. The visible difference spectrum of increasing concentration of the dye and same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum, and an isosbestic point at 569, 501, and 512 nm, respectively. The difference spectrum of the dye and dye in 60% ethylene glycol showed a maximum and a minimum at 556 and 495 nm, respectively. The dye showed no visible difference spectrum in the presence of hexokinase modified at the active site by iodoacetamide, pyridoxal phosphate, and o-phthalaldehyde. Hexokinase modified by the dye coeluted with the unmodified enzyme during size-exclusion chromatography in the absence or presence of guanidinium hydrochloride. These results suggest that the dye interacts with the hydrophobic environment of the active site of the enzyme. Analysis of the kinetics of inhibition of hexokinase by model compounds and comparison of their computer-assisted three-dimensional representations with that of Cibacron brilliant red 3B-A suggest that 1-amino-8-naphthol-3,6-disulfonic acid may represent the minimum structure for the dye to bind.

Reocclusion after thrombolytic therapy: strategies for inhibiting thrombin-induced platelet aggregation.

The use of first generation plasminogen activators, urokinase, streptokinase and tissue plasminogen activator has revolutionized thrombolytic therapy for myocardial infarction and ischaemia, and potentially stroke. However, thrombolytic therapy employing these activators is limited by reocclusion of the very arteries being opened, which follows in a small but significant number of patients. The development of second generation plasminogen activators, e.g. staphylokinase and anisoylated plasminogen streptokinase activator complex, has not alleviated the problems encountered with classical plasminogen activators. It is now widely recognized that aberrant platelet aggregation induced primarily by thrombin, rather than plasmin, is one of the major causes of recurrent thrombosis following pharmacologic thrombolysis. Agents that (a) inhibit enzymatic and/or coagulant activity of thrombin, (b) block binding of thrombin to its receptor, and (c) interfere with the generation of thrombin by the prothrombinase complex may compromise haemostasis resulting in haemorrhage. We recently demonstrated that thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a putative ADP-receptor on the platelet surface, and that these events are indirectly mediated by intracellularly activated calpain expressed on the surface. In this review, we discuss the known mechanisms of thrombin-induced platelet aggregation and suggest relative advantages of potential pharmacological agents, being developed in our laboratory, over those that have been previously developed and tested. These inhibitors selectively prevent aggregation of platelets induced by thrombin by inhibiting calpain expressed on the surface. Moreover, one of these inhibitors which blocks thrombin-induced platelet aggregation does not interfere with other platelet responses mediated by thrombin or platelet aggregation induced by other agonists, such as, ADP, collagen, phorbol myristate acetate and thromboxane A2 mimetics. This selectivity could reduce the chances of perturbing the formation of a haemostatic plug.

Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine.

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.

Thrombin- and cathepsin G-induced platelet aggregation: effect of protein kinase C inhibitors.

Two serine proteases, thrombin and cathepsin G, are potent agonists of human platelet activation. These pathophysiological proteases induce similar platelet responses, e.g., aggregation, shape change, and secretion of the dense granules. Maintenance of proteolytic function and the ability to bind to receptors on the platelet surface membrane are required for the responses elicited by both proteases. Protein kinase C (PKC) is a signal-transducing enzyme that is an important regulator of postreceptor intracellular changes following exposure of platelets to thrombin and cathepsin G. Inhibitors of purified PKC, e.g., staurosporine and calphostin C, have been frequently used to elucidate biochemical mechanisms mediated by the intracellular PKC following platelet activation by proteases. However, the effect of the PKC inhibitors on the amidolytic activity and on the ability of the two bioregulatory proteases to bind to cells has never been investigated. We found that staurosporine (1 and 1.5 microM), calphostin C (10 and 60 microM), and fisetin (200 and 220 microM), the three most commonly used and biochemically well-characterized inhibitors of purified PKC, completely inhibited thrombin-induced (2 nM) and cathepsin G-induced (0.85 microM) aggregation of washed human platelets, respectively. Each of the three PKC inhibitors completely blocked platelet shape change induced by thrombin (1 nM). Only fisetin inhibited platelet shape change induced by cathepsin G (0.5 microM). Only fisetin partially inhibited amidolytic activity of thrombin. The three PKC inhibitors had no inhibitory effect on the amidolytic activity of those concentrations of cathepsin G that cause maximum platelet aggregation and platelet shape change. The three PKC inhibitors completely blocked binding of 125I-thrombin to washed platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Aggregation of washed platelets by plasminogen and plasminogen activators is mediated by plasmin and is inhibited by a synthetic peptide disulfide.

Plasmin is known to activate platelets. However, it is not clear whether plasminogen activators as used in thrombolytic therapy can aggregate platelets and how this relates to the ability of each activator to convert plasminogen to plasmin. Urokinase (UK) and streptokinase (SK) activated purified plasminogen (2 microM) in a concentration-dependent manner. The rates of aggregation of washed platelets by the above plasminogen activators and plasminogen were similar to the extent of activation of plasminogen to plasmin in the absence of platelets. UK or SK (0.2 microM) and plasminogen (2 microM) aggregated platelets modified by an ADP affinity analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), and cleaved aggregin, a putative ADP receptor, in [3H]FSBA-modified platelets. These results suggest that the effect was independent of ADP. In contrast, incubation mixtures containing only plasminogen (2 microM) and single chain tissue plasminogen activator (sc-tPA) (less than or equal to 0.12 microM) neither activated the zymogen to an appreciable extent nor aggregated platelets. But, in the presence of fibrin(ogen) fragments (tPA-stimulator), a mixture of plasminogen and sc-tPA aggregated unmodified and FSBA-modified platelets, and cleaved aggregin. The results imply that platelets, in the presence of t-PA stimulator, potentiate activation of plasminogen to plasmin by t-PA, as previously reported. P1, Phe-Gln-Val-Val-Cys-(NpyS)-Gly-NH2, (NpyS = 3-nitro-2-thiopyridine), a synthetic hexapeptide capable of binding to and inhibiting calpain, has been shown to inhibit platelet aggregation induced by purified plasmin. P1 inhibited platelet aggregation by plasminogen and any of the three plasminogen activators. Our results show that at plasma concentrations of plasminogen and at levels of UK and SK attained after infusion of these agents during thrombolysis, these mixtures can cause maximum aggregation which may contribute to reocclusion and stenosis following infarct therapy. P1 can effectively inhibit platelet aggregation under such conditions.

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)

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

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