The fibrinogen gamma chain dodecapeptide inhibits agonist-induced aggregation of rabbit platelets and fibrinogen binding to rabbit glycoprotein IIb-IIIa

The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the y chains and the RGD sequences in the Aalpha chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen gamma chain sequence. Proteins of approximately 135 kDa and approximately 95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.     

Inhibition by ticlopidine of Paf-acether-induced in vitro aggregation of rabbit and human platelets

Ticlopidine was incubated $\text{in vitro}$ with rabbit or human washed platelets and aggregations were triggered by submaximal concentrations of adenosine-5′-diphosphate (ADP), arachidonic acid (AA) and Paf-acether (platelet-activating factor), the mediators of the three known pathways of platelet activation. Inhibition of Paf-acether-induced rabbit platelet aggregation was proportionnal to the concentrations of Ticlopidine used. The same range of inhibition by Ticlopidine was observed when aggregations were triggered by the two other agonists. Human platelet aggregation induced by Paf-acether was also inhibited by Ticlopidine. Inhibition was increased when platelets were rendered insensitive to ADP and AA. Our results show that Ticlopidine inhibits human and rabbit platelet aggregation triggered by Paf-acether through a mechanism not related to the inhibition of the ADP and prostaglandin pathways.     

Comparison of the interactions of fibrinogen and soluble fibrin with washed rabbit platelets stimulated with ADP

Because fibrin, formed at a site of vessel wall injury, is involved in the formation and stabilization of a platelet aggregate or thrombus, we have studied reactions of fibrin with rabbit platelets. Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was used to prepare soluble fibrin. Fibrin alone did not cause aggregation of washed platelets, but addition of ADP caused aggregation and deaggregation identical to those observed in the presence of fibrinogen. Specific binding of 125I-fibrin to ADP-stimulated platelets was similar to that of 125I-fibrinogen, but 125I-fibrin did not dissociate, even in the presence of high concentrations of apyrase. High non-specific binding of 125I-fibrin was observed that was not associated with aggregation. EDTA, prostaglandin E1 (PGE1) and creatine phosphate/creatine phosphokinase prevented ADP-induced aggregation in the presence of fibrin and caused rapid deaggregation when added after ADP. They also inhibited 125I-fibrin binding when added before ADP, and EDTA or PGE1 caused partial dissociation of bound 125I-fibrin. In vivo, fibrin may bind to stimulated platelets, polymerize, form a gel, and interact with components of the plasma, the platelet aggregate, and the exposed subendothelium.     

ADP receptors of platelets and their inhibition

ADP plays a crucial role in haemostasis and thrombosis and its receptors are potential targets for antithrombotic drugs. Two G-protein coupled P2 receptors contribute to platelet aggregation: the P2Y1 receptor initiates aggregation through mobilisation of calcium stores, while the more recently identified P2Y12 receptor coupled to adenylyl cyclase inhibition is essential for a full aggregation response to ADP and the stabilisation of aggregates. The latter is defective in certain patients with a selective congenital deficiency of aggregation to ADP. It is also the target of the antithrombotic drug clopidogrel and of ATP analogues and other compounds currently under evaluation. In addition, the P2X1 ionotropic receptor is present in platelets but its role is not yet completely known. Studies in P2Y1 knock-out mice and experimental thrombosis models using selective P2Y1 antagonists have shown that the P2Y1 receptor, like the P2Y12 receptor, is a potential target for new antithrombotic drugs.     

Ristocetin-induced aggregation of canine platelets

Both canine plasma and canine platelets have been reported to be unreactive in the ristocetin-induced platelet aggregation reaction that is commonly used to study the platelet-von Willebrand factor interaction. Because canine plasma was recently found to have ristocetin cofactor (RCF) activity with human platelets, we studied the RCF reactivity of canine platelets. Although canine PRP did not aggregate with ristocetin, ristocetin did induce aggregation of canine gel-filtered platelets (GFP), apparently because inhibitory plasma proteins, especially albumin, had been separated from the platelets during gel filtration. When human and canine GFP were washed, they lost their responsiveness to ristocetin. Although the ristocetin responsiveness of washed human GFP was completely restored by both human and canine factor VIII, washed canine GFP aggregated with ristocetin only in the presence of canine factor VIII. Human platelets, when fixed in paraformaldehyde aggregated well with ristocetin and either human or canine factor VIII. In contrast, paraformaldehyde-fixed canine platelets aggregated very poorly with ristocetin and factor VIII. These studies show that the ristocetin-RCF-platelet interaction functions well in the dog, although the reaction is much more species specific than the similar human reaction.     

Contribution of platelets to the cardiovascular effects of ADP in the rat

The contribution of platelets to the cardiovascular effects of ADP was investigated in rats in different experimental conditions. Following rapid i.v. bolus injections of ADP (from 0.001 to 0.03 mg/kg b.w.) only a dose-related fall in blood pressure could be detected. Increasing the dose of ADP (up to 1 mg/kg b.w.), platelet fall and changes in cardiac rhythm (bradycardia, A. V. blocks and ectopic beats) became evident. All these phenomena were rapidly reversed. Inhibition of platelet aggregation by a pyrimido-pyrimidine compound (SH 869) or thrombocytopenia induced by Busulfan or antiplatelet antiserum did not significantly protect the animals from the cardiovascular effects of ADP. The fall in blood pressure, however, was reduced. Adenosine, at aquimolar concentrations, caused ECG changes similar to those induced by ADP with no platelet aggregation and a less pronounced blood pressure fall. These results suggest that most of the cardiovascular modifications induced by rapid injection of ADP are largely independent of platelets. Platelets appeared to play a more important role when ADP was given for a longer period of time. A slow i.v. infusion of ADP (6 mg/kg b.w. for 10 min) was accompanied by platelet fall, cardiovascular collapse and ECG alterations typical of myocardial ischaemia. All these effects persisted throughout the ADP infusion but disappeared soon after its termination. They were almost completely inhibited in rats given SH 869 or made thrombocytopenic. In conclusion, platelets seem to contribute to the cardiovascular effects of ADP only in certain experimental conditions. In others, the nucleotide's effects seen more important.     

Inhibition by glaucocalyxin A of aggregation of rabbit platelets induced by ADP, arachidonic acid and platelet-activating factor, and inhibition of [3H]-PAF binding.

Glaucocalyxin A is a new diterpenoid isolated from the ethereal extract of the leaves of Rabdosia japonica (Burm f) Hara var glaucocalyx (Maxim) Hara (Labiatae) collected in the northeastern China. When it was incubated with washed rabbit platelets, glaucocalyxin A inhibited ADP- or arachidonic acid-induced platelet aggregation with IC50 values of 4.4 mumol/l, 14.1 mumol/l respectively. Glaucocalyxin A also inhibited PAF-induced aggregation of rabbit platelets which were refractory to ADP and arachidonic acid with an IC50 value of 13.7 mumol/l. Analysis of [3H]-PAF binding showed that glaucocalyxin A prevented [3H]-PAF binding to intact washed rabbit platelets with an IC50 value of 8.16 mumol/l, which was consistent with its inhibition of PAF-induced platelet aggregation.     

Shear stress-induced aggregation of oxidized platelets

Although shear stress-induced platelet aggregation (SIPA) has been noted, the shear stress-induced aggregation of oxidized platelets (SIOPA) has not been investigated. To investigate SIOPA, small quantity of hemoglobin (Hb) solution was first added to plasma to induce oxidation stress of platelets and then the plasma was sheared by a cone/plate viscometer at shear stress of 66 dyn/cm2. In addition, to investigate the change in expression of the membrane receptors of glycoprotein Ib (GpIb) for oxidized platelets, mean fluorescence intensity (MFI) for those platelets was detected by flow cytometric technique. The results showed that the level of the oxidation stress of platelets, as presented in malondialdehyde (MDA) values, was well correlated with the quantities of added Hb. Moreover, the aggregation of SIOPA corresponded with the levels of oxidation stress of platelets. Platelets pretreated with aspirin could only partially reduce the aggregation of SIOPA. Similarly, aspirin pretreatment could partially reduce the lowering of MFI value that indicated downexpression of GpIb receptors. Furthermore, the aggregations of SIOPA corresponded with the lowered percentage of those MFI values for studied cases. We thereby suggested that the aggregation of SIOPA is related to both the level of oxidation stress and the downexpression of GpIb receptors for oxidized platelets.     

Adhesion and aggregation of thrombin prestimulated human platelets on surface-bound fibrinogen: evidence for involvement of ADP and arachidonic acid pathways

The purpose of this study was to examine the influence of substances released from human platelets upon their accumulation on human fibrinogen-coated glass tubes. After prestimulation with thrombin for one minute or in the absence of prestimulation, washed human platelets suspended in Eagle's medium with RBC were drawn through the tubes at 1 ml/min, 80 s-1, for 1, 2 or 6 min. Thrombin prestimulation (0.02, 0.05 or 0.25 U/ml) was followed by inactivation with hirudin (0.1, 0.25 or 1.25 U/ml) before flow. Singly adherent platelets were observed in the absence of thrombin or with thrombin for exposure times of 1 and 2 min. At 6 min after at least 0.05 U/ml of thrombin, surface-bound aggregates were observed. The initial rate of adhesion increased with the amount of thrombin used for prestimulation. For adhesion to fibrinogen in the absence of prestimulation, platelet-derived ADP was a stimulator. Adhesion was shown to be independent of the ADP and arachidonic acid pathways in response to prestimulation with a low level of thrombin, 0.02 U/ml. For adhesion and cohesion, aggregation, in the presence of sufficient thrombin for prestimulation, 0.05 U/ml, ADP, serotonin and substances from arachidonic acid metabolism acted jointly to stimulate platelets.     

An analysis of inhibitory effect of cyclic GMP on arachidonic acid- or collagen-induced aggregation in rabbit platelets

Aggregation of washed rabbit platelets induced by arachidonic acid (AA) or collagen was inhibited by nitroprusside (NP) and 8-bromo cyclic GMP in a concentration-dependent manner. Although NP and 8-bromo cyclic GMP inhibited the AA-induced aggregation, these agents did not affect the conversion of exogenous AA to PG endoperoxides and TXA2 (which were observed as TXB2). On the other hand, collagen caused release of AA from phospholipids and sequential formation of TXB2 in [14C]AA prelabeled platelets. In contrast with the case in which exogenous AA was used, NP and 8-bromo cyclic GMP inhibited the collagen-induced formation of TXB2 by preventing the liberation of endogenous AA. These results indicate that cyclic GMP has at least two different inhibitory actions in platelets; one is the inhibition of AA release from phospholipids and the other is the inhibition of the action of TXA2 in platelets.     

Vanilloid-like agents inhibit aggregation of human platelets

Introduction

Plant-derived and endogenous vanilloid-like agents exert their effects on cells through transient receptor potential vanilloid-1 (TRPV1). Little is known about the effects of these agents on platelet aggregation. We investigated the effect of various vanilloid-like agents on in-vitro platelet aggregation and tested whether this action is mediated through TRPV1. Understanding the mechanism of action of these compounds in platelets is important in that these compounds may be developed as novel anti-platelet agents.

Materials and Methods

The effects of plant-derived (capsaicin; dihydrocapsaicin, DHC) and endogenous vanilloid-like agents (N-oleoyldopamine, OLDA; N-arachidonoyl-dopamine, NADA) on platelet aggregation were investigated using ADP (5, 10 μM), collagen (4, 8 μg/mL) and arachidonic acid (AA, 300, 400 μg/mL) as agonists. The direct effects of these agents on platelet viability were also determined using an LDH release assay.

Results

Capsaicin, OLDA and NADA inhibited ADP-induced platelet aggregation in a concentration-dependent manner. OLDA and NADA, but not capsaicin and DHC, inhibited collagen-induced aggregation, whereas AA-induced aggregation was inhibited by capsaicin, DHC and NADA, but not OLDA. Inhibition of aggregation was not due to direct toxicity of these agents towards platelets. The TRPV1 antagonist, SB-452533, did not affect inhibition of ADP-induced platelet aggregation by capsaicin and OLDA.

Conclusions

These results demonstrate that the endovanilloids, OLDA and NADA, and plant-derived vanilloid, capsaicin, inhibit ADP-induced platelet aggregation. Collagen-induced aggregation was inhibited only by endovanilloids, whereas AA-induced aggregation was inhibited by capsaicin, DHC and NADA. This inhibition was not due to direct toxic effects of these agents, nor was inhibition of ADP-induced aggregation TRPV1 mediated.     

Creatine phosphate in rat platelets decreased by ADP or thrombin

ADP or thrombin caused a transient change in creatine phosphate (CP) content of rat platelets. At 25 degrees C the CP content decreased most 30 sec after the addition of ADP or thrombin and recovered thereafter. ATP or AMP was without effect. The rat platelets aggregated at 25 degrees C by ADP or thrombin, but the changes in CP content by these inducers were also observed in the platelet preparations unable to aggregate by the lack of Ca2+. A persistent chilling made the platelets lose their ability to change CP content. It was suggested that in rat platelets CP is consumed in the early stage of the response to the aggregating agents, presumably donating the energy for shape change.     

Malondialdehyde production by platelets during secondary aggregation

A method is described for increasing the sensitivity of the thiobarbiturate assay for malondialdehyde by concentrating the coloured reaction product. The basal level of malondialdehyde-like material in plasma was found to be about 0.03 micrometer. Platelets synthesized malondialdehyde when stimulated by collagen or thrombin and also during the second phase of aggregation induced by ADP or adrenaline.     

Effects of plasmin on rabbit platelets

The effects of plasmin have been examined because platelets may be exposed to plasmin in vivo and treatment of platelets with plasmin shortens platelet survival. Rabbit plasmin was prepared by urokinase activation of plasminogen immobilized on lysine-Sepharose. Plasmin caused rabbit platelets to aggregate and release the contents of their amine storage granules, but aggregation was slower than in response to ADP or thrombin. EDTA, prostaglandin E1, or creatine phosphate/creatine phosphokinase were inhibitory, but indomethacin was not. Deaggregation did not occur when platelets had been aggregated by a concentration of plasmin that caused extensive release of granule contents. EDTA or prostaglandin E1 caused deaggregation. Low concentrations of ADP and plasmin acted synergistically in causing platelet aggregation. Plasmin decreased the amounts of platelet membrane glycoproteins that stained with periodic acid-Schiff reagent; glycoprotein I was more susceptible than glycoprotein II and III. Concentrations of plasmin that induced the release of amine storage granule contents also released PAS-staining granule glycoproteins. Platelets incubated with plasmin, washed and resuspended, were not aggregated by ADP, but were aggregated strongly by the combination of fibrinogen and ADP, and bound 125I-fibrinogen to a greater extent than untreated platelets. Platelets preincubated with a high concentration of plasmin were unresponsive to thrombin, but were sometimes aggregated by fibrinogen. Plasmin decreased the buoyant density and increased the median size of platelets. Thus plasmin, as well as ADP and thrombin, may contribute to the density shift observed in platelets from rabbits in which thrombosis and continuous vessel injury have been induced.     

Extraction of protein-bound ATP and ADP from human platelets in plasma

Actin is the major ATP and ADP binding protein in platelets, 0.9-1.3 nmol/10(8) cells, 50-70% in the unpolymerized state. The goal of these experiments was to develop a method for extracting all protein-bound ATP and ADP from undisturbed platelets in plasma. Extraction of actin-bound ADP is routine while extraction of actin-bound ATP from platelets in buffer has been unsuccessful. Prior to extraction the platelets were exposed to 14-C adenine, to label the metabolic and actin pools of ATP and ADP. The specific activity was determined from the actin-bound ADP in the 43% ethanol precipitate. Sequential ethanol and perchlorate extractions of platelet rich plasma, and the derived supernatants and precipitates were performed. ATP concentrations were determined with the luciferase assay, and radioactive nucleotides separated by TLC. A total of 1.18 nmol/10(8) cells of protein-bound ATP and ADP was recovered, 52% ATP (0.61 nmol). The recovery of protein-bound ADP was increased from 0.3 to 0.57 nmol/10(8) cells. This approach for the first time successfully recovered protein bound ATP and ADP from platelets in a concentration expected for actin.     

Kinetics of merthiolate-induced aggregation of human platelets.

Incubation of human platelet-rich plasma (PRP) or washed platelets with merthiolate (MT; sodium ethylmercurithiosalicylate; an inhibitor of lysophosphatide: arachidonoyl transferase) leads to irreversible platelet aggregation which is parallelled by an increase in thromboxane A2 synthesis. MT-induced aggregation is preceded by a pronounced lag-period (0.5-10 min). Duration of the latter is inversely related to the concentration of MT ([MT]). Platelet responses to MT are similar to those triggered by arachidonate (AA) in that the relationships of the aggregation rates both to [MT] and [AA] are threshold and exhibit characteristic super-high values of the apparent Hill coefficients (h > 30). A typical MT-induced response can be subdivided in two sequential phases: i) cyclooxygenase-independent slow aggregation, and ii) indomethacin-abrogated rapid aggregation. MT-induced responses are blocked by PGE1 or ajoene (which inhibits binding of fibrinogen to its cell surface receptor, GPIIb/IIIa). The obtained data are interpreted both quantitatively and qualitatively in terms of a model assuming the existence of: i) a relationship between the rate of MT-inhibitable AA incorporation into phospholipids and the concentration of intracellular free AA, [AA]i; ii) a certain threshold value of [AA]i essential for triggering the second phase of the aggregation.     

Competitive inhibition by lithium and hydrogen ions of the effect of calcium on the aggregation of rabbit platelets.

ADP-induced platelet aggregation and shape change were monitored optically in citrated rabbit platelet-rich plasma (PRP) diluted with isotonic salt solutions. Lithium (Li) produced a concentration-dependent reduction in the rate of platelet aggregation but had no discernible effect on the shape change which precedes aggregation. When PRP was pre-incubated with Li, the inhibitory effect of the ion was independent of the duration and temperature of the treatment. The inhibitory effect of Li also was observed in heparinized PRP or when 5-HT was used as the aggregation-inducing agent. When Li was combined with aggregation inhibitors which enhance platelet cyclic AMP content either by activating adenylate cyclase or by inhibiting phosphodiesterase, only additive effects were observed. The inhibitory effect of Li was opposed by added calcium. Kinetic evaluation of the interaction between Li and Ca indicated that their antagonism was competitive. Added calcium also displayed competitive antagonism toward the aggregation inhibiting effect of increased hydrogen ion concentration in the pH range between 6 and 8.     

An antiplatelet monoclonal antibody that inhibits ADP and epinephrine-induced aggregation

A monoclonal antibody (Mab) named EDU-3, was produced by fusing splenocytes from one Balb/c mouse, immunized with a mixture of platelets and non-T cells from heparinized human peripheral blood, with the HAT-sensitive myeloma line P3-NS1/1.Ag4.1. By indirect immunofluorescence (IF) it was seen that this Mab reacted with all normal human platelets and bone marrow megakaryocytes, but did not react with lymphoid cells from normal donors, or platelets from Glanzmann's thrombasthenia (GT) patients. Immunoprecipitation and SDS-PAGE experiments demonstrated that this Mab recognized an epitope on the IIb-IIIa glycoprotein complex (GPC). EDU-3 inhibited platelet aggregation and release of ATP induced by ADP and epinephrine. Aggregation induced by arachidonic acid, ristocetin and bovine factor VIII were not inhibited by EDU-3. The difference between EDU-3 and other Mab directed against the IIb-IIIa GPC is discussed.