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.     

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.     

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.     

Effect on perfusion chamber thrombus size in patients with atrial fibrillation during anticoagulant treatment with oral direct thrombin inhibitors, AZD0837 or ximelagatran, or with vitamin K antagonists

Introduction

AZD0837 and ximelagatran are oral direct thrombin inhibitors that are rapidly absorbed and bioconverted to their active forms, AR-H067637 and melagatran, respectively. This study investigated the antithrombotic effect of AZD0837, compared to ximelagatran and the vitamin K antagonist (VKA) phenprocoumon (Marcoumar^®), in a disease model of thrombosis in patients with non-valvular atrial fibrillation (NVAF).

Methods

Open, parallel-group studies were performed in NVAF patients treated with VKA, which was stopped aiming for an international normalized ratio (INR) of ≤ 2 before randomization. Study I: 38 patients randomized to AZD0837 (150, 250 or 350 mg) or ximelagatran 36 mg twice daily for 10-14 days. Study II: 27 patients randomized to AZD0837 250 mg twice daily or VKA titrated to an INR of 2-3 for 10-14 days. A control group of 20 healthy elderly subjects without NVAF or anticoagulant treatment was also studied. Size of thrombus formed on pig aorta strips was measured after a 5-minute perfusion at low shear rate with blood from the patient/control subject.

Results

Thrombus formation was inhibited by AZD0837 and ximelagatran. Relative to untreated patients, a 50% reduction of thrombus size was estimated at plasma concentrations of 0.6 and 0.2 μmol/L for AR-H067637 and melagatran, respectively. For patients receiving VKA treatment, the thrombus size was about 15% lower compared with healthy elderly controls.

Conclusions

Effects of AZD0837 and ximelagatran on thrombus formation were similar or greater than for VKA therapy and correlated with plasma concentrations of their active forms.     

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.     

Comparison of antithrombotic and haemorrhagic effects of edoxaban, an oral direct factor Xa inhibitor, with warfarin and enoxaparin in rats

Introduction

Factor Xa (FXa) is a key serine protease in the coagulation cascade and a promising target for a new antithrombotic agent. Edoxaban is an oral, selective and direct FXa inhibitor. The objective of this study was to compare the antithrombotic and haemorrhagic effects of edoxaban with clinically available anticoagulants, warfarin and enoxaparin, in rat models of thrombosis and haemorrhage.

Methods

Rats were treated with single oral administration of edoxaban, repeated oral dosing of warfarin for 4 days and single subcutaneous administration of enoxaparin before thrombosis or haemorrhage induction. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava for 60 min. Tail template bleeding time was measured after making an incision on the tail.

Results

Edoxaban at 0.3, 1 and 3 mg/kg exerted dose-dependent and significant inhibition of venous thrombus formation. The 50% thrombus inhibition dose (ED₅₀) was 1.9 mg/kg. At supra-therapeutic doses (10 and 20 mg/kg), edoxaban significantly but moderately (less than 2-fold) prolonged bleeding time. Warfarin and enoxaparin also dose-dependently inhibited venous thrombosis and prolonged bleeding time. The ED₅₀ values of warfarin and enoxaparin were 0.12 mg/kg and 500 IU/kg, and the 2-fold bleeding time prolongation doses (BT2) were 0.16 mg/kg and 1700 IU/kg, respectively. The safety margin (ratio of BT2 to ED₅₀) of edoxaban (> 10.5) was greater than those of warfarin (1.3) and enoxaparin (3.4).

Conclusions

Edoxaban inhibited venous thrombosis comparably to warfarin and enoxaparin, and the attendant bleeding risk of edoxaban was lower than that of warfarin and enoxaparin in rats.     

Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults

Introduction

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation.

Materials and Methods

An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated.

Results

STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (p < 0.001) and the maximum amplitudes of thrombin and plasmin generation were less in children than adults (p < 0.01). No significant differences were measured among pediatric age groups. The most profound impairments in thrombin generation were observed for extrinsic and common pathway factor deficiencies, with the exception of afibrinogenemia. Plasmin generation was severely impaired in deficiencies of fibrinogen and plasminogen as well as with decreased tPA reagent concentration and addition of aminocaproic acid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent.

Conclusion

Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults.     

Prevalence and significance of anti-prothrombin (aPT) antibodies in patients with Lupus Anticoagulant (LA)

Objective

Anti-prothrombin (aPT) antibodies have been found in Lupus Anticoagulant (LA) positive patients. Their prevalence and relative contribution to thromboembolic risk in LA-positive patients is not well defined. The aim of this study was to determine their presence and association with thromboembolic events in a large series of patients with confirmed LA.

Methods

Plasma from LA-positive patients was collected at Thrombosis Centers and sent to a reference central laboratory for confirmation. Positive plasma was tested using home-made ELISA for the presence of aPT and anti-β₂GPI antibodies.

Results

LA was confirmed in 231 patients. Sixty-one of 231 (26%, 95%CI 22-33) LA positive subjects were positive for IgG aPT and 62 (27%, 95% CI 21-33) were positive for IgM aPT antibodies. Clinical features of Antiphospholipid Syndrome (APS) were not associated with the presence of IgG aPT [43 APS in 61 (70%) positive and 109 APS in 170 (64%) negative IgG aPT subjects, p = ns] or IgM aPT. Rate of positivity of IgG and IgM aβ₂GPI was significantly higher than that of IgG and IgM aPT. Clinical events accounting for APS occurred in 97 of 130 (75%) IgG aβ₂GPI positive and in 55 of 101 (54%) IgG aβ₂GPI negative patients (OR 2.4, 95% CI 1.4 to 4.3, p = 0.002). No significant association with clinical events in patients positive for both IgG aPT and IgG aβ₂GPI as compared to those positive for one or another test was found. When patients negative for both IgG aPT and IgG aβ₂GPI (LA positive only) were compared with remaining patients, a significantly lower association with clinical events was found (OR = 0.4, 95% CI: 0.2 to 0.7, p = 0.004).

Conclusions

As compared to IgG aβ₂GPI, the prevalence of IgG aPT in patients with LA is significantly lower and not associated with the clinical features of APS.     

Evaluation of ticagrelor pharmacodynamic interactions with reversibly binding or non-reversibly binding P2Y(12) antagonists in an ex-vivo canine model

Introduction

As ticagrelor, clopidogrel and cangrelor therapies may be used in the same clinical setting, their potential pharmacodynamic interactions are of interest. Hence, we investigated possible interactions between these agents in dogs using a variety of switching protocols.

Methods

Six male dogs all received 7 different dosing regimens separated by 1-5 week washout periods: cangrelor (1 μg/kg/min, intravenous infusion); ticagrelor (0.8 mg/kg, oral); clopidogrel (3 mg/kg, intravenous injection); cangrelor together with ticagrelor initiated 10 minutes after cangrelor infusion start or clopidogrel given 30 minutes after cangrelor infusion start; ticagrelor followed by clopidogrel given 3 or 7 hours after ticagrelor dosing. ADP-induced whole blood platelet aggregation was measured by impedance aggregometry.

Results

Mean maximum inhibition of platelet aggregation (IPA) was 81-87% at 6 minutes (cangrelor), 3 hours (ticagrelor) and 4 hours (clopidogrel) postdosing and platelet function recovered after 1.5 hours, 12 hours, and 9 days, respectively. IPA at 2 hours post clopidogrel was reduced to 39% when clopidogrel was given during cangrelor infusion versus 69% for clopidogrel alone. With clopidogrel dosed 3 hours after ticagrelor, IPA was reduced after washout of ticagrelor to 38% at 24 hrs vs. 68% for clopidogrel alone, but an interaction was not seen when clopidogrel was dosed 7 hours after ticagrelor. No pharmacodynamic interaction occurred between ticagrelor and cangrelor.

Conclusions

The extent of the pharmacodynamic drug-drug interactions observed between clopidogrel and cangrelor or ticagrelor apparently depends on the level of receptor occupancy when clopidogrel is administered. Importantly, no significant pharmacodynamic interaction occurred between ticagrelor/clopidogrel when clopidogrel was given at clinical trough IPA levels with ticagrelor. No significant pharmacodynamic interaction occurred with cangrelor and ticagrelor.     

Epidemiology of venous thromboembolism in children with malignant diseases: a single-center study of the Belarusian Center for Pediatric Oncology and Hematology

Background

Venous thrombosis is a complication of treatment of children with cancer but studies devoted to the epidemiology of thrombosis in children with cancer are rare and data are scanty.

Objective

To determine the prevalence and clinical characteristics of VT as a secondary complication in children with malignant disease and to estimate the ten-year experience of our hospital.

Method

Retrospective analysis of data of Children's Cancer Subregistry of Belarus, which included information about age, gender, details of diagnosis, classification of malignant neoplasm according to ICD-10, treatment protocol and outcome. Clinical information was obtained from case histories.

Results

For the specified period, 2061 children with newly diagnosed cancer and 44 cases of VT have been registered. Among VT cases, hematological malignancies prevailed (32 of total 44). Higher incidence of VT in AML and APL groups was shown (p‹0.05). In patients with VT, boys (M/F = 1,6/1) and teenagers prevailed (65,9%). Of 44 patients, 33 had catheter-associated thrombosis (CAT). Almost all CAT (91,7%) were in the upper venous system. Children with non-CAT (11 out of 44) had more prolonged duration of immobilization, than children with CAT (p‹0.05) and in this group, thrombosis affected predominantly the lower limb (9 out of 11).

Conclusion

The present study has shown that venous thrombosis occurs significantly more often in children with AML and APL. Prevalence of boys in patients with venous thrombosis has been noted. Increased frequency of VT events in teenagers has been observed and the provoking role of CVC and immobilization for thrombosis has been confirmed.     

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.     

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.     

Elevated platelet angiostatin and circulating endothelial microfragments in idiopathic pulmonary arterial hypertension: A preliminary study

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by pulmonary arteriolar narrowing and degeneration associated with in situ thrombosis. We hypothesized that microvascular endothelial injury and apoptosis may be an initiating mechanism in IPAH. Endothelial apoptosis generates endothelial microfragments (EMF), which can activate platelets. Platelets release both VEGF and angiostatin, which depending the balance can inhibit or induce endothelial apoptosis, respectively.

Materials and Methods

We measured EMFs from blood of IPAH patients as index of endothelial cell apoptosis/injury and levels of pro- and anti- EC apoptotic factors found in platelets. EMFs and platelets in blood samples from control subjects and patients with IPAH were measured using a 4-color flow cytometry protocol, and platelet levels of VEGF and angiostatin were determined by ELISAs and immunoblotting.

Results

Compared to controls, IPAH patients exhibited higher numbers of circulating EMFs and more activated/apoptotic platelets. IPAH patients also exhibited higher levels of platelet angiostatin; however, no significant difference was detected in platelet VEGF levels between the two groups.

Conclusions

These results are consistent with an increase in EC dysfunction in patients with IPAH, possibly contributing to the progression of IPAH and its associated thrombosis.     

Sleep deprivation and accidental fall risk in children

Objectives

To look for an association between sleep deprivation and risk of accidental falls (AF) in children.

Methods

A questionnaire was applied to two groups of children aged 1-14 years, encompassing children observed in an emergency room for AF (G1) and children attending health care visits (HV) (G2). Collected data included demographic characteristics, medical history, previous week’s sleep pattern (PWSP), sleep duration and sleep pattern in the preceding 24 h, mechanism of fall, and injury severity. Exclusion criteria: acute or chronic disease or exposure to drugs interfering with sleep. Statistical analyses included Fisher’s exact test, Pearson Chi-square, Fisher-Freeman-Halton test, T and Mann-Whitney tests for independent samples, and multivariate logistic regression (α = 5%).

Results

We obtained 1756 questionnaires in G1 and 277 in G2. Of those, 834 in G1 and 267 in G2 were analyzed. We found an increased risk of AF in boys (OR 1.6; 95% CI 1.2-2.4). After controlling for age, gender, summer holidays, parental education and profession, lack of naps and PWSP were associated with increased risk (OR 2.1; 95% CI 1.3-3.3 and OR 2.7; 95% CI 1.2-6.1). In 3-5 year-old children there was an association between AF and a shorter than usual sleep duration in the previous 24 h (p = 0.02).

Conclusions

To our knowledge, our study is the largest so far to assess the association between sleep deprivation and childhood injury. It evidences a protective effect of naps in children. Sleep duration of less than 8 h increases risk of AF. Pre-schoolers may be particularly susceptible to sleep deprivation.     

Inhibition of factor XI reduces thrombus formation in rabbit jugular vein under endothelial denudation and/or blood stasis

Introduction

In addition to acquired and inherited risk factors, the growth of venous thrombus under static conditions and endothelial injury play important roles in the development of deep venous thrombosis (DVT), for which risk factors include increased plasma levels of coagulation factor XI (FXI). The aim of this study is to understand the role of FXI in venous thrombus formation under conditions of endothelial denudation and/or blood stasis.

Materials and methods

The contribution of FXI to venous thrombus formation was investigated in a rabbit model and a flow chamber system. Thrombi were induced in the rabbit jugular veins by (1) endothelial denudation, (2) vessel ligation (blood stasis) or (3) by combined endothelial denudation and vessel ligation. Blood samples were perfused on immobilized type III collagen at a wall shear rate of 70/s and then the surface area covered by platelets and fibrin was morphometrically evaluated. Prothrombin fragment 1 + 2 (F1 + 2) generation was also measured before and after perfusion.

Results

All thrombi induced in rabbit jugular veins were composed of platelets, fibrin and erythrocytes. Anti-FXI antibody significantly reduced ex vivo plasma thrombin generation initiated by ellagic acid but not by tissue factor, and in vivo thrombus formation under endothelial denudation and/or vessel ligation. The antibody significantly reduced surface areas covered by platelets and fibrin, as well as F1 + 2 generation at a wall shear rate of 70/s in flow chambers.

Conclusion

These results suggest that FXI contributes to venous thrombus growth under conditions of endothelial denudation and/or blood stasis, and that thrombin generation by FXI interaction promotes further platelet aggregation and fibrin formation at low shear rates.     

Comparative performance of warfarin pharmacogenetic algorithms in Chinese patients

Introduction

Multiple warfarin pharmacogenetic algorithms have been confirmed to predict warfarin dose more accurately than clinical algorithm or the fixed-dose approach. However, their performance has never been objectively evaluated in patients under low intensity warfarin anticoagulation, which is optimal for prevention of thromboembolism in Asian patients.

Material and methods

We sought to compare the performances of 8 eligible pharmacogenetic algorithms in a cohort of Chinese patients (n = 282) under low intensity warfarin anticoagulation with target international normalized ratio (INR) ranged from 1.6 to 2.5. The performance of each algorithm was evaluated by calculating the percentage of patients whose predicted dose fell within 20% of their actual therapeutic dose (percentage within 20%), and the mean absolute error (MAE) between each predicted dose and actual stable dose.

Results

In the entire cohort, the pharmacogenetic algorithms could predict warfarin dose with the average MAE of 0.87 ± 0.17 mg/day (0.73-1.17 mg/day), and the average percentage within 20% of 43.8% ± 8.1% (29.1% - 52.1%). By pairwise comparison, warfarin dose prediction was significantly more accurate with the algorithms derived from Asian patients (48.6% - 50.0%) than those from Caucasian patients (29.1% - 39.7%; odds ratio [OR]: 1.61-3.36, p ≤ 0.02). Algorithms with additional covariates of INR values or CYP4F2*3 performed better than those without the covariates (adding INR: OR: 1.71 (1.08-2.72), p = 0.029; adding CYP4F2*3: OR: 2.67(1.41-5.05), p = 0.004). When the patients were stratified according to the dose range, the algorithms from Caucasian and racially mixed populations tended to perform better in higher dose group (≥ 4.5 mg/day), and algorithms from Asian populations performed better in intermediate dose group (1.5-4.5 mg/day). None of the algorithms performed well in lower dose group (≤ 1.5 mg/day).

Conclusions

No eligible pharmacogenetic algorithm could perform the best for all dosing range in the Chinese patients under low intensity warfarin anticoagulation. Construction of a refinement pharmacogenetic algorithm integrating 3 genotypes (CYP2C9, VKORC1 and CYP4F2) and INR data should be warranted to improve the warfarin dose prediction in such patients.     

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.     

Circulating platelet-derived microparticles and endothelium-derived microparticles may be a potential cause of microthrombosis in patients with osteonecrosis of the femoral head

Introduction

To test the hypothesis that the platelet microparticle (PMP) and endothelial microparticle (EMP) may contribute to the hypercoagulability associated with microvascular thrombosis in patients with nontraumatc osteonecrosis of the femoral head (ONFH).

Materials and methods

The study comprised 46 patients who had been diagnosed with ONFH and 20 control subjects. The plasma was ultracentrifuged, and then PMPs and EMPs were examined by the flow cytometry. The thrombotic and fibrinolytic disorders were investigated.

Results

The numbers of PMPs expressing P-selectin and CD42a and EMPs expressing E-selectin and CD31 in the ONFH patients were significantly higher than those in the controls (P < 0.001). The number of MPs was correlated with the level of the serum C-reactive protein (CRP) (r = 0.661, P < 0.001), but there was a poor correlation between the MPs counts and the risk factors for ONFH (P > 0.05). The mean levels PAI-1, F1 + 2, and TAT were higher in the patients with ONFH than in the controls (P < 0.05).

Conclusions

The elevated numbers of PMPs and EMPs may contribute to hypercoagulability in the ONFH patients. This may provide important pathophysiological insights into the hypercoagulability associated with nontraumatic ONFH and have implications for pharmacological prevention and treatment of ONFH.     

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model

Introduction

Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.

Materials and methods

The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF^flox/floxLysMCre⁺ mice that have reduced TF expression in myeloid cells, (2) TF^flox/floxLysMCre^- littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.

Results

Inhibition of TF significantly decreased thrombus weight 2 days post venous thrombosis. In contrast, TF^flox/floxLysMCre⁺ had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2 days but then returned to baseline levels by 6 days post thrombosis. D-dimer levels peaked at 2 days post thrombosis in mice with or without myeloid cell TF.

Conclusions

TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.     

Parameters of the thrombogram are associated with serum 25-hydroxyvitamin D levels at baseline, but not affected during supplementation with vitamin D

Introduction

In vitro studies indicate an anticoagulant effect of 1,25-dihydroxyvitamin D, and sun exposure may lower the risk of thrombotic events. Accordingly, an effect on haemostatic parameters could be expected after supplementation with vitamin D.

Materials and Methods

158 obese or overweight subjects were included in a one year intervention study with supplementation with 40.000 IU vitamin D₃ per week or placebo. All subjects were given 500 mg calcium daily. Plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator antigen (tPA Ag), and tissue factor-induced thrombin generation over time in plasma assessed by the calibrated automated thrombogram (CAT) method as a parameter of over all thrombotic activity, were measured before and at the end of the study.

Results

Mean baseline serum 25(OH)D level was 61.8 nmol/L and increased in the vitamin D group to 145.6 nmol/L at the end of the study. At baseline there was a significant decrease in the CAT variables lag time and time to peak of the thrombogram across increasing serum 25(OH)D quartiles, whereas no significant associations between serum 25(OH)D and PAI-1 or tPA Ag were found. After one year, no significant differences were found between the vitamin D and placebo groups regarding change in any of the haemostatic parameters.

Conclusions

The association between lag time and time to peak in the CAT assay and serum 25(OH)D levels could indicate a pro-thrombotic state in subjects with high serum 25(OH)D levels, whereas the lack of effect of high dose vitamin D supplementation questions the causality of this relation.