I(4), a new synthetic sulfonylurea compound, inhibits the action of TXA(2)in vivo and in vitro on platelets and aorta vascular smooth muscle

Introduction

1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl]phenylsulfonyl]-3-(trans-4-methylcy-clohexyl)urea(I₄, CAS865483-06-3); a totally synthetic new sulfonylurea compound, combining the hypoglycemic active structure of Glimepiride (CAS 93479-97-1) and anti-TXA₂ receptor (TP) active structure of BM-531(CAS 284464-46-6), was designed and synthesized. Its effects on TXA₂ synthesis and TP have not been reported yet.

Aim

To study the inhibitory effects of I₄ and its mechanisms of action on TXA₂ and TP.

Methods

Platelet aggregation studies were performed on human platelet, rat whole blood platelet and rabbit platelet, platelets aggregation was induced by TP agonist U-46619(stable analog of TXA₂, CAS 56985-40-1). Plasma TXB₂ and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were used as markers to determine the effect of I₄ on thromboxane synthesis. Fluo-3-AM was used to measure the cytosolic Ca^2 + concentrations ([Ca^2 +]_i) in rabbit platelet. Aorta rings with and without endothelium were prepared and aorta contraction was induced by U-46619. A model of type 2 diabetes mellitus was established by intraperitoneal injection of low dose of streptozocin to rats fed a high-calorie diet. Both normal rats and type 2 diabetic rats were used to assay the inhibitory effect of I₄ on platelet aggregation induced by U-46619.

Results

I₄ exhibited a higher inhibitory potency than Glimepiride on U-46619 induced platelet aggregation in vitro and in vivo. I₄ increased the ratio of plasma PGI₂/TXA₂ and decreased [Ca^2 +]_i release from platelet internal stores. In addition, I₄ presented a vasorelaxant activity on isolated rat aorta contraction induced by U-46619.Oral administration of I₄ (1 ~ 10 mg/kg) markedly and dose-dependently inhibited platelet aggregation in both normal rats and type 2 diabetic rats.

Conclusion

I₄ significantly inhibited platelet aggregation induced by U-46619 in vitro and in vivo, and rat aorta contraction. It probably acts by partly blocking TXA₂ action, decreasing the platelet intracellular Ca^2 +, and increasing the PGI₂/TXA₂ ratio.     

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.     

Decichine enhances hemostasis of activated platelets via AMPA receptors

Introduction

Dencichine, one of the non-protein amino acids present in the roots of Panax notoginseng, has been found to shorten bleeding time of mice and increase the number of platelets. However, the exact underlying mechanisms have not been elucidated yet. This study was aimed to identify the hemostatic effect of dencichine and uncover its mechanisms.

Materials and methods

Hemostatic effect was assessed by measuring tail bleeding time and coagulation indices of rats. PT, APTT, TT and FIB concentration were measured using a Sysmex CA-1500 plasma coagulation analyzer. Platelet aggregation rate was determined by using a platelet aggregometer. Concentration of cyotosolic calcium was evaluated by Fluo-3 and levels of cyclic adenosine monophosphate (cAMP) and thromboxane A₂ (TXA₂) were measured by ELISA method.

Results and conclusion

Dencichine administered orally shortened tail bleeding time, reduced APTT and TT but increased the concentration of FIB in plasma in a dose-dependent manner. When induced with trap, dencichine could elevate the cytoplasmic concentration of calcium, and secretion of TXA₂ as well as the ratio of TXA₂ to PGI₂ from platelets. Meanwhile, it decreased the level of intracellular cAMP. However, CNQX could block the enhanced hemostatic effect of dencichine. These results suggested that dencichine exerted hemostatic function via AMPA receptors on platelets, therefore, facilitated coagulation cascade in a paracrine fashion by control of platelet cytosolic calcium influx, cAMP production and TXA₂ release. Current study may contribute to its clinical use in therapy of hemorrhage.     

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.     

A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation

Introduction

Prasugrel, a P2Y₁₂ adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADP-mediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD.

Materials and Methods

Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B₂ (TXB₂), P-selectin, and TF.

Results

Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB₂ were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects.

Conclusions

These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD.     

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction

Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects.

Materials and Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs.

Conclusions

CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.     

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.     

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.     

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.     

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.     

Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates

Introduction

Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied.

Method

Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined.

Results

Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6 mg/kg, significantly attenuated thrombus formation.

Conclusions

Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent.     

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.     

2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) attenuates human platelet aggregation,secretion and spreading in vitro

Introduction

2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside(THSG) is a water-soluble component of the rhizome extract from the traditional Chinese herb Polygonum multiflorum. Recent studies have demonstrated that THSG has potent anti-oxidative and anti-inflammatory effects. In this study, we investigated the anti-platelet aggregation, secretion and spreading of THSG with different methods. The purpose was to explore the anti-platelet effect of THSG and the underlying mechanism.

Materials and Methods

We investigated the anti-platelet activity of THSG on platelet aggregation induced by collagen (2 μg/mL), thrombin(0.04U/mL), U46619 (3 μM) and ADP (2 μM). ATP secretion induced by collagen (2 μg/mL) was also investigated. P-selectin expression and PAC-1 binding were measured by flow cytometry. In addition, human platelet spreading on immobilized fibrinogen and immunoblotting were also tested.

Results

THSG dose-dependently inhibited platelet aggregation and ATP secretion induced by collagen. It inhibited platelet P-selectin expression and PAC-1 binding induced by thrombin(0.1U/mL). THSG also inhibited human platelet spreading on immobilized fibrinogen, a process mediated by platelet outside-in signaling. Western blot analysis showed that THSG could inhibit platelet Fc γ RIIa, Akt(Ser473)and GSK3β(Ser9) phosphorylation.

Conclusions

Our study indicates that THSG has potent anti-platelet activity to collagen induced aggregation. THSG is likely to exert protective effects in platelet-associated thromboembolic disorders by modulating human platelet.     

Mice with deleted multimerin 1 and α-synuclein genes have impaired platelet adhesion and impaired thrombus formation that is corrected by multimerin 1

Background

Multimerin 1 is a stored platelet and endothelial cell adhesive protein that shows significant conservation. In vitro, multimerin 1 supports platelet adhesion and it also binds to collagen and enhances von Willebrand factor-dependent platelet adhesion to collagen. As selective, multimerin 1 deficient mice have not been generated, we investigated multimerin 1 effects on platelet adhesion using a subpopulation of C57BL/6J mice with tandem deletion of the genes for multimerin 1 and α-synuclein, a protein that inhibits α-granule release in vitro. We postulated that multimerin 1/α-synuclein deficient mice might show impaired platelet adhesive function from multimerin 1 deficiency and increased α-granule release from α-synuclein deficiency.

Methods

Platelet function was assessed by intravital microscopy, after ferric chloride injury, using untreated and human multimerin 1-transfused multimerin 1/α-synuclein deficient mice, and by in vitro assays of adhesion, aggregation and thrombin-induced P-selectin release.

Results

Multimerin 1/α-synuclein deficient mice showed impaired platelet adhesion and their defective thrombus formation at sites of vessel injury improved with multimerin 1 transfusion. Although multimerin 1/α-synuclein deficient platelets showed increased P-selectin release at low thrombin concentrations, they also showed impaired adhesion to collagen, and attenuated aggregation with thrombin, that improved with added multimerin 1.

Conclusions

Our data suggest that multimerin 1 supports platelet adhesive functions and thrombus formation, which will be important to verify by generating and testing selective multimerin 1 deficient mice.     

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.     

Molecular Cloning and Characterization of Rhesus Monkey Platelet Glycoprotein Ibα, a major ligand-binding subunit of GPIb-IX-V complex

Introduction

Through binding to von Willebrand factor (VWF), platelet glycoprotein (GP) Ibα, the major ligand-binding subunit of the GPIb-IX-V complex, initiates platelet adhesion and aggregation in response to exposed VWF or elevated fluid-shear stress. There is little data regarding non-human primate platelet GPIbα. This study cloned and characterized rhesus monkey (Macaca Mullatta) platelet GPIbα.

Materials and Methods

DNAMAN software was used for sequence analysis and alignment. N/O-glycosylation sites and 3-D structure modelling were predicted by online OGPET v1.0, NetOGlyc 1.0 Server and SWISS-MODEL, respectively. Platelet function was evaluated by ADP- or ristocetin-induced platelet aggregation.

Results

Rhesus monkey GPIbα contains 2,268 nucleotides with an open reading frame encoding 755 amino acids. Rhesus monkey GPIbα nucleotide and protein sequences share 93.27% and 89.20% homology respectively, with human. Sequences encoding the leucine-rich repeats of rhesus monkey GPIbα share strong similarity with human, whereas PEST sequences and N/O-glycosylated residues vary. The GPIbα−binding residues for thrombin, filamin A and 14-3-3ζ are highly conserved between rhesus monkey and human. Platelet function analysis revealed monkey and human platelets respond similarly to ADP, but rhesus monkey platelets failed to respond to low doses of ristocetin where human platelets achieved 76% aggregation. However, monkey platelets aggregated in response to higher ristocetin doses.

Conclusions

Monkey GPIbα shares strong homology with human GPIbα, however there are some differences in rhesus monkey platelet activation through GPIbα engagement, which need to be considered when using rhesus monkey platelet to investigate platelet GPIbα function.     

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.