MC-002 exhibits positive effects against platelets aggregation and endothelial dysfunction through thromboxane A2 inhibition

Introduction

Thromboxane A₂ (TXA₂) induces platelet aggregation and vasoconstriction, and agents that inhibit TXA₂ production or interaction with receptors may exert potential application in stroke therapy.

Aim

To illustrate the platelet aggregation antagonistic and endothelial protective effect of (E) - 3 - (3 - methoxy - 4 - ((3, 5, 6 - trimethylpyrazin - 2 - yl) methoxy) phenyl) sodium acrylate (MC-002) through TXA₂ inhibition and underline mechanisms.

Materials and methods

Platelets aggregation and thoracic aorta ring contraction of rabbits were induced by U46619. Human umbilical vein endothelial cells (HUVECs) were further applied to explore the protective effect of MC-002 on endothelium when exposed to tumor necrosis factor - α (TNF-α). MTT method was used to assess cell damage, and ELISA analysis was exerted to estimate nitrogen monoxide (NO), endothelin-1 (ET-1), thromboxane B₂ (TXB₂) and 6-keto-prostaglandin F1α (6-keto-PGF1α) releasing. Fluorescence spectrophotometry was conducted to determine intracellular calcium concentration ([Ca^2 +]_i), and western blotting method was applied to evaluate the protein expressions of intracellular adhesion molecule-1 (ICAM-1), P-selectin and nuclear factor-kappa B (NF-κB).

Results and conclusions

TXA₂ analog U46619 mediated obvious platelet aggregation and vasoconstriction. MC-002 inhibited platelet aggregation through administration in vivo and incubation with platelet in vitro, and relaxed aorta ring in endothelium dependent manner. MC-002 alleviated cell damage, [Ca^2 +]_i overload, ET-1 overexcretion and TXB₂ activation, but improved NO availability reduction in HUVECs treated with TNF-α. Furthermore, MC-002 downregulated ICAM-1, P-selectin and NF-κB overexpression induced by TNF-α. In conclusion, MC-002 exerted antiplatelet aggregation effect through TXA₂ inhibition and relieved inflammatory injury of endothelial cells through NF-κB signal pathway.     

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets

Introduction

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A₂ (TxA₂) receptor.

Material and methods

Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA₂ receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA₂ was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA₂ mimic) and the calcium ionophore A23187.

Results

The data showed that aglycone isoflavones and equol bind to TxA₂ receptor in the µmol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [³H]-SQ29585 binding was: equol > genistein > daidzein > glycitein ? genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA₂ binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA₂ analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP.

Conclusions

The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A₂ receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.     

ASP6537, a novel highly selective cyclooxygenase-1 inhibitor,exerts potent antithrombotic effect without “aspirin dilemma”

Introduction

Aspirin inhibits both the cyclooxygenase (COX)-1-dependent production of thromboxane A₂ (TXA₂) in platelets and COX-2-dependent production of anti-aggregatory prostaglandin I₂ (PGI₂) in vessel walls, resulting in “aspirin dilemma.” Our objective is to investigate whether ASP6537 can overcome aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect.

Methods

We evaluated the inhibitory effects of ASP6537 on recombinant human COX-1 (rhCOX-1) and rhCOX-2 activities using a COX-1/2 selectivity test. To determine whether ASP6537 induces aspirin dilemma, we examined the effects of ASP6537 on in vitro TXA₂ and PGI₂ metabolite production from platelets and isolated aorta of guinea pigs, and on plasma concentrations of TXA₂ and PGI₂ metabolites in aged rats. Finally, we evaluated the antithrombotic effects and ulcerogenic activity of ASP6537 using an electrically induced carotid arterial thrombosis model and a gastric ulcer model in guinea pigs.

Results

The IC₅₀ ratios of rhCOX-2 to rhCOX-1 for ASP6537 and aspirin were > 142,000 and 1.63 fold, respectively. ASP6537 inhibited TXA₂ production more selectively than aspirin in in vitro and in vivo TXA₂/PGI₂ production studies. ASP6537 exerted a significant antithrombotic effect at ≥ 3 mg/kg, while aspirin tended to inhibit thrombosis at 300 mg/kg but it was not statistically significant. Further, ASP6537 did not induce ulcer formation at 100 mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of ≥ 100 mg/kg.

Conclusions

ASP6537 functions as a highly selective COX-1 inhibitor with a superior ability to aspirin for normalizing TXA₂/PGI₂ balance, and exerts antithrombotic effect without ulcerogenic effect.     

Reduced platelet response to aspirin in patients with coronary artery disease and type 2 diabetes mellitus

Introduction

Diabetes mellitus is complicated by accelerated atherosclerosis, resulting in an increased risk of coronary artery disease (CAD) and thrombosis. Despite the proven benefits of aspirin, previous studies indicate a reduced cardiovascular protection from aspirin in diabetic patients. We aimed to investigate whether diabetes mellitus influenced the platelet response to aspirin in patients with CAD.

Materials and Methods

Platelet aggregation and activation were evaluated during aspirin treatment in 85 diabetic and 92 non-diabetic patients with CAD. Adherence to aspirin was carefully controlled. All patients had CAD verified by coronary angiography and were taking 75 mg non-enteric coated aspirin daily.

Results

Diabetic patients showed significantly higher levels of platelet aggregation compared to non-diabetic patients evaluated by VerifyNow^® Aspirin (p = 0.03) and Multiplate^® aggregometry using arachidonic acid (AA) 0.5 mM (p = 0.005) and 1.0 mM (p = 0.009). In addition, platelet activation determined by soluble P-selectin was significantly higher in diabetics compared to non-diabetics (p = 0.005). The higher AA-induced aggregation was associated with higher levels of HbA_1c. Compliance was confirmed by low levels of serum thromboxane B₂ (below 7.2 ng/mL). Diabetics had significantly higher levels of serum thromboxane B₂ (p < 0.0001).

Conclusions

Diabetic patients with CAD had significantly higher levels of both platelet aggregation and activation compared to non-diabetic patients with CAD despite treatment with the same dosage of aspirin. These findings may partly explain the reduced cardiovascular protection from aspirin in diabetic patients.     

Altered platelet calsequestrin abundance,Na/Ca exchange and Ca signaling responses with the progression of diabetes mellitus

Introduction

Downregulation of calsequestrin (CSQ), a major Ca^2 + storage protein, may contribute significantly to the hyperactivity of internal Ca^2 + ([Ca^2 +]_i) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca^2 + signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca^2 + influx in diabetic platelets.

Materials and methods

Type 1 diabetes was induced by streptozotocin in rats. Platelet [Ca^2 +]_i and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting.

Results

During the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca^2 +]_i and ADP-induced Ca^2 + release were progressively altered in diabetic platelets, while the elevated Ca^2 + influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca^2 + channel blocker, almost completely abolished ADP-induced Ca^2 + influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na⁺/Ca^2 + exchange induced much slower Ca^2 + extrusion and more Ca^2 + influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca^2 +-ATPase inhibition, ionomycin caused greater Ca^2 + mobilization and Ca^2 + influx in diabetic platelets than in normal platelets.

Conclusions

These data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca^2 + homeostasis, insufficient Na⁺/Ca^2 + exchange also contributes, at least in part, to the hyperactive Ca^2 + response to stimulation in diabetic platelets.     

Platelet aggregometry in the presence of PGE(1) provides a reliable method for cilostazol monitoring

Introduction

Cilostazol has been shown to be effective for prevention and treatment of cerebral infarction. However, there appears to be no widely accepted method appropriate for monitoring cilostazol. We attempted to establish an assay system for cilostazol monitoring, using platelet aggregation induced by arachidonic acid (AA) in the presence of PGE₁ which upregulates intracellular cyclic AMP.

Methods

Blood was drawn from stroke patients before and after cilostazol intake. AA-induced platelet aggregation after pretreatment with 0 ~ 30 nM PGE₁ for 2 minutes was measured by light transmittance aggregometry.

Results

AA-induced platelet aggregation was 73.1 ± 2.2% in the absence of PGE₁, and pretreatment with 30 nM PGE₁ had virtually no inhibitory effect on platelet aggregation prior to cilostazol intake. In contrast, after cilostazol intake, 30 nM PGE₁ significantly inhibited platelet aggregation to 12.7 ± 4.5% (p = 7.8 × 10(− 11)) , while in the absence of PGE₁ platelet aggregation remained similar to that of prior-to-cilostazol value (70.6 ± 3.5%). The plasma concentration of cilostazol ranged from 0.55 to 3.51 μM. In the presence of 30 nM PGE₁, all the patients with cilostazol concentrations exceeding 1 μM had their platelet aggregation inhibited almost completely. ROC analysis suggests that AA-induced platelet aggregation in the presence of 30 nM PGE₁ had the excellent sensitivity (90.5%) and specificity (88.4%) for monitoring cilostazol.

Conclusions

AA-induced platelet aggregation in the presence of 30 nM PGE₁ could give good estimate on plasma concentrations of cilostazol. It is suggested that this system is a good tool for monitoring cilostazol.     

The prevalence of the platelet glycoprotein IIIa Pl(A1/A2) polymorphism in three South African ethnic groups and its effect on platelet function

Introduction

In South Africa coronary artery disease (CAD) is less common in African than Indian or white subjects. Although the association between CAD and metabolic factors have been well documented, the role of genetic factors is as yet poorly understood. Specific polymorphisms in the platelet membrane glycoprotein (GP) IIIa gene Pl^A1/A2, have been implicated in the development of CAD.

Methods

The prevalence of platelet GPIIIa (Pl^A1/A2) polymorphisms and their effect on platelet function was determined in 313 Indian, 267 white and 227 African subjects with and without a history of CAD.

Results

In subjects without a history of CAD the frequency of the unfavourable Pl^A2 allele was 8.0%, 14.8% and 8.7% in the Indian, white and African populations respectively, with the frequency being significantly higher (p < 0.05) in the white than both other groups. The frequency of the Pl^A2 allele was higher in subjects with (23.0%) than without (10.0%; p < 0.0001) a history of CAD. Aggregation studies showed that platelets carrying the Pl^A2 allele were hypersensitive to the platelet aggregating agonists ADP and collagen and produced a higher amount of TXA₂ when stimulated with low concentrations of both these agonists.

Conclusions

The positive association observed between the platelet GPIIIa Pl^A1/A2 polymorphism and platelet function suggests that the GPIIIa Pl^A2 allele may be a genetic factor that contributes to the risk of sudden death from myocardial infarction in the absence of known risk factors but it does not explain ethnic differences in the prevalence of CAD.     

Aspirin treatment influences platelet-related inflammatory biomarkers in healthy individuals but not in acute stroke patients

Objectives

Platelet-leukocyte aggregation is believed to contribute to acute thrombotic events. While the effect of aspirin on platelet-to-platelet aggregation is well established, the impact of the drug on pro-inflammatory platelet function remains equivocal. Thus we investigated the effect of aspirin on selected platelet-related inflammatory biomarkers in both acute ischaemic stroke patients and healthy volunteers.

Methods

Using five-colour flow cytometry the platelet surface expression of CD62P and CD40L and subpopulations of leukocyte-platelet aggregates were assessed in 63 acute stroke patients and 40 healthy volunteers at baseline and after a 10-day period of aspirin intake at a daily dose of 150 mg. Simultaneously the plasma levels of soluble CD62P and CD40L, serum level of TxB₂, and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were investigated.

Results

No differences in values of studied platelet-related inflammatory biomarkers in both resting platelets and those activated with TRAP after 10-day treatment with aspirin were confirmed in stroke subjects. In healthy individuals the resting platelet expression of CD62P, plasma level of soluble CD62P and percentage of circulating monocyte-platelet aggregates were lower after the aspirin intake period (P = 0.009; P = 0.04; P = 0.004, respectively). In both studied groups serum level of TxB₂ and platelet aggregation under AA stimulation were lower than before treatment (P < 0.001).

Conclusion

Despite effective inhibition of COX-1-dependent platelet aggregation, aspirin does not influence the platelet α-granule-derived inflammatory mediators and monocyte-platelet aggregation in acute stroke subjects, although it does in healthy individuals.     

Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis

Introduction

Derived from the root of Panax ginseng C.A.Mey, Panax notoginsenosides (PNS) is a widely used herbal medicine to treat atherothrombotic diseases in Asian medicine. Ginsenoside Rg1 is one of the main compounds responsible for the pharmaceutical actions of PNS. As platelets play pivotal roles in atherothrombogenesis, we therefore studied the effect of Rg1 on platelet activation and its underlying mechanisms.

Materials and Methods

Human platelets are obtained from healthy subjects. Platelet activation and the inhibition of Rg1 were assessed by Born aggregometer, flow cytmetry, flow chamber and western blot. The in vivo thrombosis model was induced by 10% FeCl₃ on mesenteric arterioles of wild type B57/b6 mice.

Results

Rg1 significantly inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, e.g., aggregation rate stimulated by 0.1 U mL^- 1 thrombin was decreased 46% by Rg1. Rg1 also reduced thrombin (0.1 U mL^- 1)-enhanced fibrinogen binding and P-selectin expression of single platelet by 81% and 66%, respectively. Rg1 affected αIIbβ3-mediated outside-in signaling as demonstrated by diminished platelet spreading on immobilized fibrinogen. Rg1 also decreased the rate of clot retraction in platelet rich plasma. Furthermore, Rg1 decreased platelet adhesion on collagen surface under a shear rate correlated to the arterial flow (1000 s^- 1) by approximately 70%. Western blot showed that Rg1 potently inhibited ERK phosphrylation. The in vitro findings were further evaluated in the mouse model of in vivo arterial thrombosis, and Rg1 was found to prolong the mesenteric arterial occlusion time (34.9 ± 4.1 min without and 64.3 ± 4.9 min with Rg1; p < 0.01).

Conclusions

Rg1 inhibits platelet activation via the inhibition of ERK pathway, and attenuates arterial thrombus formation in vivo.     

PGE2 decreases reactivity of human platelets by activating EP2 and EP4

Introduction

Platelet hyperreactivity associates with cardiovascular events in humans. Studies in mice and humans suggest that prostaglandin E₂ (PGE₂) regulates platelet activation. In mice, activation of the PGE₂ receptor subtype 3 (EP3) promotes thrombosis, but the significance of EP3 in humans is less well understood.

Objectives

To characterize the regulation of thromboxane-dependent human platelet activation by PGE₂.

Patients/Methods

Platelets collected from nineteen healthy adults were studied using an agonist of the thromboxane receptor (U46,619), PGE₂, and selective agonists and/or antagonists of the EP receptor subtypes. Platelet activation was assayed by (1) optical aggregometry, (2) measurement of dense granule release, and (3) single-platelet counting.

Results

Healthy volunteers demonstrated significant interindividual variation in platelet response to PGE₂. PGE₂ completely inhibited U46,619-induced platelet aggregation and ATP release in 26% of subjects; the remaining 74% had partial or no response to PGE₂. Antagonism of EP4 abolished the inhibitory effect of PGE₂. In all volunteers, a selective EP2 agonist inhibited U46,619-induced aggregation. Furthermore, the selective EP3 antagonist DG-041 converted all PGE₂ nonresponders to full responders.

Conclusions

There is significant interindividual variation of platelet response to PGE₂ in humans. The balance between EP2, EP3, and EP4 activation determines its net effect. PGE₂ can prevent thromboxane-induced platelet aggregation in an EP4-dependent manner. EP3 antagonism converts platelets of nonresponders to a PGE₂-responsive phenotype. These data suggest that therapeutic targeting of EP pathways may have cardiovascular benefit by decreasing platelet reactivity.     

Evaluation of laboratory methods routinely used to detect the effect of aspirin against new reference methods

Background

Aspirin, a commonly used antiplatelet agent, blocks platelet thromboxane A₂ (TXA₂) formation from arachidonic acid (AA) by acetylating platelet cyclooxygenase-1 (COX-1). Laboratory methods currently used to detect this antiplatelet effect of aspirin provide variable results. We have reported three methods that assess platelet COX-1 acetylation (inactivation) by aspirin and its direct consequences. The first and second assays use monoclonal anti-human-COX-1 antibodies that only detect acetylated (inactivated) COX-1 and active (non-acetylated) COX-1, respectively. The third method measures platelet production of TXB₂ (the stable metabolite of TXA₂) in vitro in response to AA. We compared the results of these three reference methods with other routinely used methods for assessing the functional consequences aspirin treatment.

Methods

108 healthy volunteers were treated with low-dose aspirin for 7 days. On day 7 following aspirin treatment COX-1 in the platelets was fully acetylated whereas only non-acetylated COX-1 was present in the day 0 platelets. Further, TXB₂ production by day 7 platelets was completely blocked. The following tests were performed on the samples obtained from study participants before and after seven days of aspirin treatment: PFA-100 closure time with collagen/epinephrine cartridge, VerifyNow® (VN) Aspirin Assay, platelet aggregation and ATP secretion using AA, ADP, epinephrine and collagen as agonists.

Results

Comparing the pre-treatment and day 7 values, methods that use AA as platelet agonist (AA-induced platelet aggregation/secretion and VN Aspirin Assay) showed high discriminative power. In contrast, results of the other tests showed considerable overlap between day 7 and day 0 values.

Conclusions

Only assays that clearly distinguish between acetylated and non-acetylated platelet COX-1 are useful for establishing the antiplatelet effect of aspirin. The other tests are not suitable for this purpose.     

Effect of P2Y1 and P2Y12 genetic polymorphisms on the ADP-induced platelet aggregation in a Korean population

Background

P2Y₁ and P2Y₁₂ receptors are expressed in platelet membranes and are involved in ADP-induced platelet aggregation. Genetic polymorphisms of P2Y₁ and P2Y₁₂ play a major role in the variation of ADP-induced platelet aggregation and in response in antiplatelet therapy.

Objective

To evaluate the allele frequencies of P2Y₁ and P2Y₁₂ genetic polymorphisms in a Korean population and to assess their role in ADP (5 μmol/L)-induced maximal platelet aggregation.

Methods

P2Y₁ (c.1622A > G) and P2Y₁₂ (i-139C > T, i-744 T > C, i-ins801, c.52G > T, c.34C > T) polymorphisms were analyzed in 158 Korean healthy participants using pyrosequencing methods. Their ADP-induced maximal platelet aggregation was assessed by the turbidometric method.

Results

The observed allele frequencies of P2Y₁ and P2Y₁₂ were as follows: 0.3101 for P2Y₁ c.1622A > G; 0.1804 for P2Y₁₂ i-139C > T, 0.1804 for i-744 T > C, 0.1804 for i-801insA, 0.1266 for P2Y₁₂ c.52G > T, and 0.2658 for P2Y₁₂ c.34C > T. ADP-induced maximal platelet aggregation was not influenced by the P2Y₁ c.1622A > G polymorphism and was also not affected by three intronic P2Y₁₂ polymorphisms and the P2Y₁₂ c.34C > T polymorphism. However, the P2Y₁₂ c.52G > T polymorphism caused a substantial difference in ADP-induced maximal platelet aggregation (62.75% for c.52GG, 66.27% for c.52GT, and 80.60% for c.52TT; P = 0.0092).

Conclusions

The P2Y₁ and P2Y₁₂ genes were very polymorphic in a Korean population. Three intronic P2Y₁₂ polymorphisms (i-139C > T, i-744 T > C, i-801insA) were in complete linkage disequilibrium but not with the c.52C > T polymorphism in this population. Maximal platelet aggregation in response to ADP is associated with the c.52C > T polymorphism but not with the three intronic polymorphisms or the P2Y₁ c.1622A > T polymorphism.     

Inhibition of platelet aggregation by curdione from Curcuma wenyujin essential Oil

Introduction

Curdione, one of the major sesquiterpene compounds from Rhizoma Curcumae, has been shown to exhibit multiple bioactive properties. In this study, we investigated the anti-platelet aggregation and antithrombotic activities of curdione with different methods both in vitro and in vivo. The purpose of the study was to explore an inhibitor of platelet aggregation, which promised to be a preventive or therapeutic agent for various vascular diseases.

Materials and Methods

Curdione was isolated from the essential oil of Curcuma wenyujin using the silica gel column chromatography method. The effects of curdione on human platelet aggregation induced by thrombin (0.3 U/ml), platelet-activating factor (PAF, 0.375 μg/ml), adenosine diphosphate (ADP, 10 μM) and arachidonic acid (AA, 0.1 mg/ml) were tested in vitro, and the potential mechanisms underlying such activities were investigated. We also tested the antithrombotic effect of curdione in a tail thrombosis model.

Results and conclusions

Curdione preferentially inhibited PAF- and thrombin- induced platelet aggregation in a concentration-dependent manner (IC₅₀: 60-80 μM), whereas much higher concentrations of curdione were required to inhibit platelet aggregation induced by ADP and AA. Curdione also inhibited P-selectin expression in PAF-activated platelets. Moreover, curdione caused an increase in cAMP levels and attenuated intracellular Ca²⁺ mobilization in PAF-activated platelets. In vivo, we also found that curdione showed significant antithrombotic activity. Therefore, we conclude that the inhibitory mechanism of curdione on platelet aggregation may increase cAMP levels and subsequently inhibit intracellular Ca²⁺ mobilization. Furthermore, the effect observed in the tail thrombosis model might be explained completely by increased vasodilation. These results indicate that curdione may be one of the main bioactive constituents in Rhizoma Curcumae that removes blood stasis.     

Aspirin resistance following pediatric cardiac surgery

Introduction

Aspirin is often used to prevent thrombosis in pediatric cardiac surgery. The primary study aim was to assess aspirin resistance in this context. Secondary aims were to evaluate (1) the relationship between elevated inflammatory markers and thrombosis and (2) aspirin's effect on these levels.

Materials and Methods

This was a prospective observational study of children undergoing cardiac surgery managed with and without aspirin. Aspirin response was assessed using the VerifyNow™ system and urinary 11-dehydrothromboxane B₂ (uTxB₂) measurements. Laboratory studies of inflammation were also obtained.

Results

101 subjects were studied; 50 received aspirin. Six subjects (5.9%), 5 aspirin-treated, experienced symptomatic thrombosis. When measured by VerifyNow™ resistance was 43% after aspirin suppositories and 14% after additional days of oral aspirin. There was no correlation with thrombosis. Upper quartile post-operative day (POD) #5 uTxB₂ was correlated with thrombosis in aspirin treated subjects (p < 0.01). High risk aspirin-treated subjects who experienced thrombosis had higher POD#5 uTxB_2. This finding did not reach statistical significance (p = 0.07). Elevated pre-operative C-reactive protein (CRP) was independently associated with thrombosis (p < 0.02) in all subjects and in high risk subjects (p = 0.01). Inflammatory markers were not affected by aspirin.

Conclusions

Aspirin inhibited ex-vivo platelet function with a low incidence of resistance. Elevated POD#5 uTxB₂ and pre-operative CRP were correlated with thrombosis in aspirin treated subjects. Further studies are needed to determine whether children with high levels of uTxB₂ despite aspirin therapy and/or those with elevated preoperative CRP are at increased risk for thrombosis.     

TXA2 synthesis and COX1-independent platelet reactivity in aspirin-treated patients soon after acute cerebral stroke or transient ischaemic attack

Introduction

The pharmacological target of aspirin is the inhibition of cyclooxygenase-1 (COX1) and thromboxane-A₂ (TX) synthesis. Very few data are available on TX assessment in patients with stroke. We studied platelet TX synthesis, COX1-independent platelet reactivity, the influence of platelet–erythrocyte interactions and the potential association between platelet responses and the severity of stroke, evaluated with a clinical score (NIHSS).

Material and Methods

We examined 157 aspirin-treated patients with acute stroke or TIA, 128 aspirin-free and 15 aspirin-treated healthy subjects (HS). Collagen-induced TX, platelet recruitment in whole blood and platelets ± erythrocytes (haematocrit 40%) were assessed in patients on daily-aspirin within three days from onset. Arachidonic-acid-, ADP-, thrombin-receptor activating peptide TRAP-, and collagen-induced aggregation were also evaluated.

Results

Partial TX inhibition (< 95% inhibition vs aspirin-free controls) was observed in 13% of patients. This was associated with marked increases in COX1-dependent responses (arachidonic-acid- and collagen-induced aggregation and platelet recruitment; P < 0.0001) but not with differences in ADP- or TRAP-induced aggregation. Partial TX inhibition was independently associated with severe stroke (NIHSS ≥ 12) at both admission (P < 0.05) and discharge (P < 0.05). Among patients with fully blocked TX, those with elevated COX1-independent platelet reactivity (mean + 2SD of aspirin-treated HS) were most likely to suffer severe stroke (P < 0.05). Platelet–erythrocyte interactions enhanced platelet reactivity in these patients by COX1-dependent and -independent mechanisms (P < 0.0001).

Conclusions

TX inhibition by aspirin varied across patients. Partial TX inhibition and COX1-independent platelet hyperfunction were associated with more-severe stroke.     

Anopheline anti-platelet protein from a malaria vector mosquito has anti-thrombotic effects in vivo without compromising hemostasis

Introduction

The saliva of blood-feeding animals (e.g., mosquitoes, ticks, bats) has pharmacological activities that facilitate efficient blood-sucking. We previously identified a unique anti-platelet protein, anopheline anti-platelet protein (AAPP), from the salivary gland of female Anopheles stephensi (human malaria vector mosquito). AAPP specifically blocks platelet adhesion to collagen by binding directly to collagen and subsequently aggregating platelets. To examine the potential of AAPP as a therapeutic agent, we investigated the in vivo anti-thrombotic effects of AAPP.

Materials and Methods

Effects of AAPP on whole blood/platelet aggregation in mice were examined. AAPP was also challenged in an established model of pulmonary thromboembolism in mice. We simultaneously investigated the side-effects of the protein (prolongation of bleeding time and coagulation time). Aspirin was used as a positive control for comparison of anti-thrombotic effects.

Results and Conclusions

AAPP inhibited whole blood aggregation induced by collagen at 10 mg/kg body weight. AAPP prevented pulmonary death at a lower dose (3 mg/kg) without prolongation of bleeding time compared with aspirin (100 mg/kg) that compromised hemostasis. AAPP and aspirin did not affect coagulation time. These results indicate that AAPP has great potential as a new anti-platelet agent with a better risk/benefit ratio than that seen with aspirin (the most widely used anti-platelet agent).     

Antiplatelet and antithrombotic activities of salvianolic acid A

Introduction

Salvianolic acid A (SAA), the water-soluble phenolic acids in Salvia miltiorrhiza, has shown the most potent bioactivities, including protection against cerebral lesion, defense from oxidative damage and improvement of remembrance. In the present study, we studied the antiplatelet and antithrombotic effects of a newly synthesized SAA with different methods both in vitro and in vivo.

Materials and Methods

We tested the effect of antithrombotic activity of SAA in arterio-venous shunt model. The effects of SAA on adenosine diphosphate (ADP)-, Thrombin-, Arachidonic acid- induced rat platelets aggregation were tested both in vivo and in vitro. The activity of SAA on washed human platelet aggregation was determined by ADP stimulation. We also evaluated its property of modulation of hemorheology, assessed its bleeding side effect by measuring coagulation parameters after intravenous administration for 5 days and investigated the potential mechanisms underlying such activities.

Results and Conclusions

In vivo, SAA significantly reduced thrombus weight in the model of arterio-venous shunt. Meanwhile, SAA increased plasma cAMP level determined by radioimmunoassay in the same model. Intravenously administrated SAA (2.5-10 mg/kg) inhibited platelet aggregation induced by ADP in a dose-dependent manner. Notably, SAA did not affect coagulation parameters in rats after intravenous administration SAA for successive 5 days. In vitro, pretreatment with SAA on washed rat and human platelets significantly inhibited various agonists stimulated platelet aggregation and caused an increase in cAMP level in platelets activated by ADP. These findings support our hypothesis that SAA possesses antithrombotic activities. The antithrombotic effect might be related to its antiplatelet action and ability to modulate hemorheology without affecting coagulation system. The mechanisms underlying such activities may involve the induction of cAMP.     

A comparative study of antithrombotic and antiplatelet activities of different fucoidans from Laminaria japonica

Introduction

Fucoidans extracted from brown algae represent an intriguing group of natural fucose-enriched sulfated polysaccharide, with excellent anticoagulant, antimetastatic, antiangiogenic and anti-inflammatory activities. In the present study, we compared antithrombotic activities of four fucoidan fractions with different molecular weight and sulfated ester content from Laminaria japonica in an electrical induced arterial thrombosis and their potential mechanism underlying such activity.

Results and Conclusions

In vivo middle molecular weight (MMW) fucoidan fractions with molecular weight about 28000 and 35000 exhibited better antithrombotic activity in electrical induced arterial thrombosis than low molecular weight (LMW) fucoidan LF1 and LF2 (Mw 7600 and 3900). Inhibition of arterial thrombosis occurred at dose of 0.1-0.25 mg/kg for MMW fucoidans, accompanied with moderate anticoagulant activity and significant decrease of whole blood viscosity and hematocrit. The antithrombotic effects of MMW Fucoidans might be related with promotion of TFPI content and decrease of TXB2 content, without affecting platelet aggregation and 6-keto-PGF1α content in vivo. In contrast, LMW fucoidans showed a correlation among anticoagulant, antiplatelet and antithrombotic effects in vivo. Antithrombotic action of LF1 and LF2 required high dose of 2.5-10 mg/kg, concomitantly with anticoagulant activity and specific inhibition of platelet aggregation in vivo. Their antithrombotic effect might be related to their promotion of TFPI and 6-keto-PGF1α, down regulation of TXB2, without affecting hemorheology. These findings suggested that fucoidan fractions with different molecular weight acted on the antithrombotic action by different mechanism. By comparison, highly sulfated fucoidan LF2 with molecular weight of 3900 seemed to be a more suitable choice of antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.     

The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca signaling pathways

Introduction

Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated.

Methods

The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry.

Results

PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca^2 + mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca^2 + chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it.

Conclusion

The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca^2 + signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.     

Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP

Introduction

CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation.

Methods

The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study.

Results

CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B₂ (TxB₂) and hydroxyl radical (OH^●) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB₂ formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation.

Conclusion

This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and MAPKs), ultimately inhibiting platelet activation. This novel role of CME-1 indicates that CME-1 exhibits high potential for application in treating and preventing CVDs.