Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of ³H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.     

Prostacyclin release from the coronary vascular wall by vasoactive substances

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI₂) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 μM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 μM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 μM acetylcholine, 25 μM noradrenaline, 25 μM isoproterenol, 10 μM adenosine triphosphate (ATP 5 μM adenosine, 1 μM angiotensin II, 25 μM histamine or 1 μM serotonin was infused for 30 sec, no inhibitory activity of platele aggregation was observed. Bradykinin (5 × 10⁻⁹ 5 × 10⁻⁶ M) and purified thrombin (1 × 10⁻⁹ 1 × 10⁻⁷ M) induced a dose-dependent release of PGI₂ which was assayed using a radioimmunoassay for 6-keto-prostaglandin F₁ (6-keto-PGF₁).     

Characterization of phospholipase A2 secretion from human platelets

Human platelets secreted phospholipase A₂ in a dose- and time-dependent manner when challenged with thrombin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or collagen. Enzyme release was maximal at concentrations of 0.1 units/ml of thrombin, 100 nM TPA, or 2 μg/ml of collagen; and complete by 2 min in platelets treated with thrombin or TPA. Cells challenged with collagen required up to 5 min for maximal secretion. Besides dose and time functions, phospholipase A₂ secretion was also dependent on platelet concentration and the levels of bovine serum albumin in the incubation medium. The secreted enzyme was soluble and exhibited substrate and Ca²⁺ requirements similar to a detergent-solubilized, partially purified phospholipase A₂ from whole platelets [Kramer et al., Biochim. Biophys. Acta (1988) 959, 269–279]. The pH optimum of the secreted enzyme, however, was 1–2 units lower than the pH optimum of the phospholipase A₂ from whole cells. Secreted phospholipase A₂ hydrolyzed phosphatidylethanolamine at 5–12 times the rate of phosphatidylcholine when the substrates were present in pure form. These apparent differences in activity were greatly diminished, though, when 1:1 molar mixtures of the two substrates were employed. Because phospholipase A₂ catalyzes a key reaction during the formation of bioactive arachidonate metabolites, the secretion of this enzyme from platelets may be important in the regulation of thrombosis.     

Platelet aggregation studies in whole human blood

Since the development of the photometric aggregometer, platelet aggregation studies are routinely performed on platelet-rich plasma (PRP). We have studied platelet aggregation in fresh citrated whole human blood using the recently developed Ultra Flo 100 Whole Blood Platelet Counter. Aggregation of platelets in whole blood was induced with adenosine 5′-diphosphate (ADP; 0.25–10μM), collagen (0.25–1.0μg/ml) and thrombin (0.05–0.2U/ml). Platelet aggregation induced by ADP and thrombin was maximum within 1 min, and that of collagen within 3 mins. Aggregation responses to low concentration of ADP and thrombin were rapidly reversible, whereas responses to collagen and high concentrations of ADP and thrombin were virtually irreversible. The aggregation of platelets was indicated by a fall in platelet count; confirmed by scanning electron micrographs which revealed the presence of large aggregates of platelets, and was prevented when blood was treated with EDTA as anticoagulant. The present technique appears to be rapid, sensitive and reliable; and allows direct measurement of platelet aggregation and disaggregation in whole blood in vitro and ex-vivo.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

Gly-pro-arg-pro (GPRP) enhances free thrombin

Fibrinogen-related peptides, which inhibit the interaction of fibrinogen with the platelet membrane glycoprotein IIb/IIIa complex (GPIIb/IIIa) are under preclinical investigation now (1). Whereas peptides containing the Arg-Gly-Asp (RGD) sequence inhibit fibrinogen-dependent platelet aggregation by direct binding to GPIIb/IIIa (2), GPRP inhibits fibrinogen polymerisation by direct binding to the fibrinogen polymerisation sites and modifying the glutamine residues in the - and γ-chains of fibrinogen (3, 4). Since GPRP has been shown to inhibit ADP-induced platelet aggregation it has been suggested as antithrombotic agent (5).

It has been demonstrated that in defibrinated plasma the amount of free thrombin generated after clotting activation is significantly higher than in normal plasma (6). The explanation for this observation is that in normal plasma free thrombin is partially adsorbed on generated fibrin (7). Since GPRP inhibits fibrinogen polymerisation, we investigated the generation of thrombin in platelet-rich plasma (PRP) containing GPRP.     

Some properties of the human platelet vasopressin receptor

Aggregation of human platelets by vasopressin is potently inhibited by 1-[βmercapto-(β,β′-cyclopentamethylene propionic acid)]-L-arginine vasopressin, a selective vasopressor (V₁) antagonist. 1-Desamino-8-D-arginine-vasopressin, a selective anti-diuretic (V₂) agonist failed to induce aggregation and acted as a weak antagonist. Vasopressin analogues which lacked the N-terminal amino group or which contained an uncharged amino acid residue at position 8 acted as partial agonists for the human platelet. The response to such partial agonists could be enhanced by increasing the cytosolic Ca²⁺ concentration but not by altering the level of cyclic-3′,5′-AMP. These observations provide further evidence indicating that the platelet vasopressin receptor is of the V₁ sub-type.     

Endothelin and platelet function

The effects of newly discovered vasoconstrictor peptide endothelin was studied on human, rabbit and canine platelet function. Endothelin (0.01 nM - lμM) did not promote platelet aggregation. In human platelets, endothelin (0.1 μM) did not significantly affect aggregation responses to ADP, collagen, epinephrine, arachidonic acid, PGH₂ or thrombin. Endothelin did not promote the mobilization of intracellular calcium in Fura2 loaded human platelets. In rabbit and canine platelets endothelin produced signficant potentiation of platelet aggregation mediated by low concentrations of ADP. Aggregation responses to higher concentration of ADP (5 μM) were unaffected by endothelin. These data reveal that under certain circumstances endothelin may potentiate rabbit and canine platelet aggregation responses to ADP, however endothelin does not produce direct effects on human platelet function.     

Effect of op 1206, a prostaglandin E1 derivative, on guinea-pig platelet functions

OP 1206, 17(S)-methyl-ω-homo-trans-Δ²-prostaglandin E₁, inhibited guinea-pig platelet aggregation induced by ADP, collagen, A 23187, arachidonic acid, labile aggregation stimulating substances (LASS) and thromboxane A₂ (TXA₂)-like substance, and platelet adhesiveness to a glass bead column. The potency of inhibition was 10–16 times stronger than that of prostaglandin E₁ (PGE₁). ADP-induced platelet aggregation was notably disaggregated by the addition of OP 1206 after induction of aggregation. The release of ATP and ADP from guinea-pig platelets induced by collagen was suppressed by OP 1206, of which potency was 9–10 times stronger than that of PGE₁. OP 1206 and PGE₁ increased guinea-pig platelet cyclic AMP levels, and the increased levels were augmented by the pretreatment with theophylline. OP 1206 and PGE₁ inhibited synthesis of guinea-pig platelet malondialdehyde (MDA) induced by thrombin but not by arachidonic acid. This inhibition was released by exogenous calcium. OP 1206 and PGE₁ showed no influence on synthesis of radioactive TXA₂ (measured as a stable form, TXB₂) from [¹⁴C] arachidonic acid. From these results, increased levels of platelet cyclic AMP by OP 1206 as well as PGE₁ may exert their action on platelet functions.     

Preoptic formation of 17β-oestradiol is influenced by behavioural stimuli in the dove

In vitro study of testosterone (T) metabolism shows that formation of 17β-oestradiol (E₂) in the preoptic area is higher in male doves exposed to behavioural stimuli from interacting pairs than in visually isolated males or males that have interacted with females. Formation of 5-dihydrotestosterone, 5β-dihydrotestosterone and 5β-androstane-3,17β-diol was similar in all groups, indicating that exposure to stimuli from interacting pairs influences aromatization of T and not other pathways of androgen metabolism. The results suggests that sexual stimuli, which do not involve tactile cotact between male and female, affect local E₂ formation in the brain.     

Antiplatelet effect of capsaicin

Capsaicin was found to be a potent inhibitor of platelet aggregation and release reaction. It inhibited the aggregation of rat platelet induced by collagen and thrombin, but only slightly reduced those of AA and A23187. The IC₅₀ on collagen-induced platelet aggregation was about 85 μg/ml. Less inhibition was observed in the aggregation of platelet-rich plasma. Increase of the calcium concentration could not overcome the inhibitory effect. Washing of the capsaicin-pretreated platelets only partially reversed the inhibition. Capsaicin also inhibited ATP release induced by thrombin and A23187 in the presence of EDTA. MDA and TXB₂ formation were markedly inhibited by capsaicin in platelets challenged by collagen, thrombin and A23187. In AA-stimulated platelets, MDA formation was slightly decreased and TXB₂ formation was not affected. Capsaicin showed more marked inhibition in the presence of CP/CPK, indomethacin or a combination of both. Capsaicin reduced the hemolysis of RBCs induced by hydrogen peroxide or hypotonicity. It was concluded that capsaicin had some membrane stabilizing property and this might lead to the interferance of the activation of phospholipase A₂.     

Beta 2 glycoprotein I--function in health and disease

Beta-2 glycoprotein I (β₂GPI) is the principal target of autoantibodies in the antiphospholipid syndrome (APS). It is abundant in human plasma and shares high homology between different mammalian species. Although the exact physiological function of β₂GPI has not been fully elucidated, several interactions have been described with other proteins and with negatively charged surfaces, such as anionic phospholipids, dextran and heparin. β₂GPI is involved in the coagulation pathway, exerting both procoagulant and anticoagulant activities. Plasma from β₂GPI-deficient mice exhibits impaired thrombin generation in vitro. Recently, it has been demonstrated that β₂GPI binds factor (F) XI in vitro at concentrations lower than those of the protein in human plasma, and this binding inhibits FXI activation to FXIa by thrombin and FXIIa. Proteolytic cleavage of the fifth domain of β₂GPI abolishes its inhibition of FXI activation and results in reduced ability of the cleaved β₂GPI to bind phospholipids. It retains its ability to bind FXI. In vivo activation of FXI by thrombin is thought to be an important mechanism by which coagulation is accelerated via components of the contact activation pathway. Thus β₂GPI may attenuate the contact activation pathway by inhibiting activation of FXI by thrombin. Moreover, because β₂GPI is the dominant autoantigen in patients with APS, dysregulation of this pathway by autoantibodies may be an important mechanism for thrombosis in patients with APS.     

Developing Peptide Inhibitors to Thrombin Activation of Platelets from Bradykinin Analogs

Investigations identified peptide, platelet-selective thrombin inhibitors. Three peptides (MAP4- , and ) were relatively selective inhibitors of thrombin-induced platelet activation and calcium mobilization. MAP4- at 35.5±0.03 μM inhibits γ-thrombin-induced platelet aggregation 100% and -thrombin-induced calcium mobilization in fibroblasts 84%. at 800±400 μM inhibits γ-thrombin-induced platelet aggregation 100% and calcium mobilization 63%. at 140±100 μM inhibits γ-thrombin-induced platelet aggregation 100% and calcium mobilization 32%. also inhibits ADP-induced platelet aggregation, whereas MAP4- and do not. prolongs the activated partial thromboplastin time (APTT) but not the prothrombin time (PT) or thrombin clotting time (TCT). uniquely inhibits -thrombin activation of human factor XI. Single amino acid substitutions in peptide pentamers result in differences in potency and mechanism(s) of inhibition of platelet and fibroblast activation by thrombin.     

Binding of [3H]-dihydroalprenolol and [3H]-acetobutolol to human blood platelets is not related to occupancy of beta-adrenoceptors

Binding of {³H}-dihydroalprenolol to human platelet lysates is inhibited by (±)-propranolol and (±)-butoxamine, but less effectively by (±) practolol. (−)-Isoprenaline causes no significant inhibition of binding where stimulation of adenylate cyclase can be shown. Binding of {³H}-acetobutolol is also inhibited by (±)-propranolol. “Specific” binding of {³H}-dihydroalprenolol and {³H}-acetobutolol defined by (±) propranolol shows a non-classical saturation curve. 50% maximal binding is observed in the range 15 – 25 mM. The extent of “specific” binding is 2-fold greater for {³H}-dihydroalprenolol. Similar and rapid rates of binding of {³H}-dihydroalprenolol are observed at 4°C and 20°C. No stereoselectivity is observed for inhibition of {³H}-dihydroalprenolol binding by (+) and (−)-propranolol. Binding of {³H}-dihydroalprenolol and {³H}-acetobutolol may relate to the lipophilic character of these radioligands and does not represent interaction with β-adrenoceptors.     

The release of B beta 1-42 from fibrinogen and fibrin by plasmin

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH₂-terminal end of the A chain, and of Bβ 1–42 (thrombin-increasable fibrinopeptide B immunoreactivity - TIFPB) which reflect plasmin action on the NH₂-terminal end of the Bβ chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Trisbuffered saline at pH 7.4 and at 37°C. With fibrinogen as substrate the K_m was calculated to be 0.87 μM and the V_max 3.75 × 10⁻⁵ M/min/unit of plasmin. With fibrin I monomer as the substrate the K_m was calculated to be 1.25 μM and the V_max 5.5 × 10⁻⁵ M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH₂-terminal end of the Bβ chain.     

Hypercholesterolemia-induced Abeta accumulation in rabbit brain is associated with alteration in IGF-1 signaling

Hypercholesterolemia increases levels of β-amyloid (Aβ), a peptide that accumulates in Alzheimer's disease brains. Because cholesterol in the blood does not cross the blood brain barrier (BBB), the link between circulating cholesterol and Aβ accumulation is not understood. In contrast to cholesterol, the oxidized cholesterol metabolite 27-hydroxycholesterol can cross the BBB, potentially increasing Aβ levels. However, the mechanisms by which cholesterol or 27-hydroxycholesterol regulate Aβ levels are not known. The insulin-like growth factor-1 (IGF-1) regulates the glycogen-synthase kinase-3α (GSK-3α) and the insulin degrading enzyme (IDE). While GSK-3α increases Aβ production, IDE is a major Aβ-degrading enzyme. We report here that feeding rabbits with a cholesterol-enriched diet increases Aβ levels in the hippocampus, an effect that is associated with reduced IGF-1 levels. 27-hydroxycholesterol also increases Aβ and reduces IGF-1 levels in organotypic hippocampal slices from adult rabbits. We suggest that hypercholesterolemia-induced Aβ accumulation may be mediated by 27-hydroxycholesterol, involving IGF-1 signaling.     

Differential involvement of membrane (Na-K)atpase in thrombin- and trypsin-mediated platelet activation

The possibility that platelet activation may also involve membrane (Na-K)ATPase was investigated by testing the effects of both proteinases on platelet shape change and aggregation in the absence and presence of the specific (Na-K)ATPase inhibitor ouabain. Ouabain (8 to 80 μM) completely antagonized trypsin-induced platelet shape change and aggregation when it was preincubated with platelet suspension before the addition of trypsin. Unlike trypsin, thrombin-induced platelet activation was significantly enhanced by ouabain. It was also observed that on partially purified beef heart (Na-K)ATPase preparation, thrombin significantly enhanced the enzyme inhibition caused by submaximal inhibitory concentrations of ouabain.

Soybean trypsin inhibitor (4 μg/ml) employed as the agent capable to counteract proteinase effects on the (Na-K)ATPase, was shown both to prevent and antagonize the platelet activation induced by trypsin (0.3 to 1.5 μg/ml), but it failed to modify the responses evoked by thrombin.

It is concluded that membrane (Na-K)ATPase is involved differently in platelet activation by trypsin and thrombin probably because receptor sites to which either proteinase on the platelet surface binds, are distinct. Direct enzyme involvement is indeed apparent only in trypsin-induced platelet activation.     

Prior simvastatin treatment is associated with reduced thrombin generation and platelet activation in patients with acute ST-segment elevation myocardial infarction

Background

It has been reported that statin therapy produces additional effects including impaired activation of blood coagulation. It is not clear whether statins can affect hemostasis in patients with acute coronary syndrome. The aim of this study was to investigate the effect of prior statin treatment on thrombin generation and platelet activation in patients with ST-segment elevation myocardial infarction (STEMI).

Methods

We studied 53 consecutive STEMI patients admitted within 12 hours of pain onset, including 19 treated with simvastatin (40 mg/d) (simvastatin group) at least one month prior to STEMI, and 34 not receiving any statins (no-statin group) on admission. Thrombin-antithrombin (TAT) complexes generation and soluble CD40 ligand (sCD40L) release were determined in 60-second blood samples collected at the site of microvascular injury.

Results

There were no significant intergroup differences with respect to clinical and laboratory variables, including plasma TAT and sCD40L levels, except lower total cholesterol and low-density lipoprotein cholesterol in the simvastatin group. The mean maximum rate of TAT generation was 47.5% lower (p = 0.0002) and sCD40L release 33.3% lower (p = 0.0006) in the simvastatin group. Total amounts of TAT (p < 0.0001) and sCD40L (p = 0.002) collected within 5 minutes of bleeding were lower in simvastatin-pretreated patients. By multivariate regression analysis, variables describing local TAT and sCD40L profiles in the whole group were independently associated with simvastatin pretreatment (p < 0.0001), but not with cholesterol, platelet count or troponin levels.

Conclusions

Prior simvastatin use is associated with lower thrombin generation and platelet activation following vascular injury in the early phase of STEMI.     

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E₁ (PGE₁) on platelets is mediated through the PGE₁ receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE₁ and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 gm. The time course of the extension length (Dp, in μg) of the platelets in response to aspiration with a negative pressure (ΔP) of 5 cm H₂ O (ΔP × Rp = 0.15 dynes/cm) was analyzed. PGE₁ treatment (0.1 μM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE₁ -treated) / Dp/Rp (control), was significantly reduced to 0.90 ± 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE₁ -treated platelets had a higher [cAMP]_i than controls. Platelets treated with PGE₁ or dbcAMP showed a reduced [Ca²⁺]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE₁ and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]_i and low [Ca²⁺]_i after PGE₁ treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.     

Platelet aggregation following exposure of phospholipase-treated platelet rich plasma to hydrogen peroxide

Hydrogen peroxide at micromolar concentrations (250–500 μM) can induce platelet aggregation of phospholipase-treated PRP. This effect, which occurs independently of the release of ADP, is blocked by aspirin, furosemide, catalase and 2-mercaptoethanol. PRP preincubated with H₂O₂ for 2–5 min. does not respond to Phl-H₂O₂ or collagen. This inhibitory effect is abolished with longer preincubations. In the presence of ADP or epinephrine, H₂O₂ enhances aggregation, if added to PRP with the inducers, and decreases the platelet response to the inducers, if preincubated with PRP for 2 min. The data suggest that micromolar concentrations of H₂O₂, which could be generated at sites of platelet plug formation by granulocytes, could influence the processes of hemostasis and thrombosis.