Aging is associated with impaired thrombus resolution in a mouse model of stasis induced thrombosis

Introduction

To evaluate the effects of aging on venous thrombosis.

Material and Methods

Anesthetized male mice (C57BL/6, n = 125) underwent complete inferior vena cava occlusion to produce venous thrombosis. Experimental groups included 11-month-old mice (OLD), 2-month-old mice (YOUNG), and age-matched non-thrombosed controls. Mice were euthanized and the following parameters were evaluated two days post-thrombosis: thrombus mass (grams/cm), vein wall inflammatory cells (cells per 5 high powered fields), active plasma plasminogen activator inhibitor-1 (PAI-1, ng/mL), vein wall P-selectin protein determination by ELISA (pg/mL), circulating plasma microparticles (MPs, MPs/200 µL), MP tissue factor (TF) activity (pM), and in vivo MP re-injection experiments.

Results

Thrombosed OLD mice had greater thrombus mass than YOUNG mice (389 ± 18 vs. 336 ± 14 g × 10^− 4/cm, P < .05). OLD mice had decreased vein wall monocyte, lymphocyte, and total inflammatory cell populations versus YOUNG mice (P < .05). Vein wall P-selectin levels were greater in OLD thrombosed mice versus YOUNG (7306 ± 938 vs. 3805 ± 745 pg/mL, P < .05). Active plasma PAI-1 concentrations were increased in OLD mice versus YOUNG thrombosed animals (20 ± 4 vs. 8 ± 2 ng/mL, P < .05). OLD mice had significantly higher circulating leukocyte-derived MPs versus YOUNG mice (5817 ± 850 vs. 2563 ± 283 MPs/200 μL PPP, P < .01). OLD mice had plasma MPs with increased TF activity versus YOUNG animals post-thrombosis (34 ± 4 vs. 24 ± 2 pM, P < .05). Finally, YOUNG recipient animals, whether re-injected with OLD or YOUNG donor MPs, had a significant increase in thrombus mass versus OLD recipient animals (P < .01).

Conclusion

Aging influenced several circulating and vein wall factors that decreased thrombus resolution in older animals compared to younger ones in our mouse thrombosis model.     

Microscopic clot fragment evidence of biochemo-mechanical degradation effects in thrombolysis

Introduction

Although fibrinolytic treatment has been used for decades, the interactions between the biochemical mechanisms and the mechanical forces of the streaming blood remain incompletely understood. Analysis of the blood clot surface in vitro was employed to study the concomitant effect of blood plasma flow and recombinant tissue plasminogen activator (rt-PA) on the degradation of retracted, non-occlusive blood clots. Our hypothesis was that a faster tangential plasma flow removed larger fragments and resulted in faster overall thrombolysis.

Materials and Methods

Retracted model blood clots were prepared in an optical microscopy chamber and connected to an artificial perfusion system with either no-flow, or plasma flow with a velocity of 3 cm/s or 30 cm/s with or without added rt-PA at 2 µg/ml. The clot surface was dynamically imaged by an optical microscope for 30 min with 15 s intervals.

Results

The clot fragments removed during rt-PA mediated thrombolysis ranged in size from that of a single red blood cell to large agglomerates composed of more than a thousand red blood cells bound together by partly degraded fibrin. The average and the largest discrete clot area change between images in adjacent time frames were significantly higher with the faster flow than with the slow flow (14,000 μm² and 160,000 μm² vs. 2200 μm² and 10,600 μm²).

Conclusions

On the micrometer scale, thrombolysis consists of sequential removal of clot fragments from the clot surface. With increasing tangential plasma flow velocity, the size of the clot fragments and the overall rate of thrombolysis increases.     

Interictal alterations of cytokines and leukocytes in patients with active epilepsy

Background

Involvement of the innate immune system in the pathogenesis of epilepsies has been suggested but possible interactions between the immune system and human epilepsy remain unclear. We analyzed the interictal immuno-phenotype of leukocyte subsets and proinflammatory cytokine profiles in epileptic patients and correlated them with the epilepsy syndrome.

Methods

101 patients with active focal or generalized epilepsy were prospectively included and compared to 36 healthy controls. Immuno-phenotype of leukocyte subsets and cytokines IL-1β, IL-6 and tnfα were measured in peripheral blood. Multivariate analyses were performed to test group differences.

Results

As compared to controls, the patients showed an elevated percentage of monocytes (18.06 ± 7.08% vs. 12.68 ± 4.55%, p < 0.001), NK cells (14.88 ± 7.08% vs. 11.43 ± 5.41%, p = 0.019) and IL-6 concentration (3.33 ± 3.11 pg/ml vs. 1.5 ± 1.36 pg/ml, p = 0.002). This remained true when focal epilepsies or generalized epilepsies were compared separately to controls but only focal epilepsies showed additionally a decrease in B lymphocyts (8.16 ± 3.76% vs. 11.54 ± 4.2%, p < 0.001). Treatment with lamotrigine was associated with a higher percentage of B lymphocytes and valproate with an increased percentage of CD4⁺ T lymphocytes. Therapy with levetiracetam showed a trend towards decreased CD8⁺ T cell counts. No significant differences were seen between focal and generalized epilepsies and between temporal and extratemporal lobe epilepsies.

Conclusion

Patients with active epilepsy revealed interictal alterations of the immune system which varied among specific syndromes and were influenced by antiepileptic drug treatment.     

Assessment of a cohort of primarily pediatric patients with a presumptive diagnosis of type 1 von Willebrand disease with a novel high shear rate, non-citrated blood flow device

Background

A precise approach to the diagnosis of von Willebrand disease (vWD) remains elusive. One important reason is that vWD is a blood flow‐related disorder: a vW Factor‐platelet GPIb binding defect exists in this condition under the high shear‐rate (> 1000 sec‐1 in whole blood; > 3000 sec‐1 in PRP) conditions of physiologic blood flow which exist in the arterioles of mucous membranes, from which most bleeding in vWD occurs.

Methods

We therefore studied 28 patients (mean 18.9 yrs) with vWD, diagnosed according to the 2007 NHLBI clinical guidelines, and 26 healthy controls (mean 17.5 yrs). Blood was collected into a plastic tube containing 4 U/ml FC dalteparin, 1.75 μg/ml of the Tab (anti‐CD41) monoclonal antibody directed against platelet GPIIb, and 1.0 μg/ml of an ALEXA 555‐conjugated rabbit anti‐mouse second antibody. Within 30-90 min, the blood was then withdrawn at 667 and 1330 sec^− 1 through a special flow chamber allowing for real-time epifluorescence digital videomicroscopy of platelets interacting with a microfibrillar collagen substrate. With MetaMorph software (Universal Imaging) we quantified the percent area (PA) covered by and total volume (TV) of adherent platelet aggregates within a 435 μm × 580 μm field of view.

Results

At 667 sec^− 1 after 1 min PA and TV were similar for patients and controls, but at 1330 sec^− 1 PA was 9.32 ± 4.21 (mean ± SD) for patients, a value lower (p < 0.001) than the 12.8 ± 3.39 for controls. TV was (1.43 ± 0.91)x10⁴ for patients, a value also lower (p < 0.001) than the (2.22 ± 0.77)x10⁴ for controls. PA or TV was below the 2.5th percentile for controls in 10 patients (36%) and both PA and TV were below the 2.5th percentile in eight.

Conclusions

The novel flow device found that PA and TV were significantly reduced under high shear stress in vWD patients compared to normal controls. However, there was some overlap between the vWD and the control group, suggesting that some vWD patients had normal platelet adhesion/aggregation under the conditions studied. Further study with a higher shear rate appears indicated.     

Partial versus complete factor VIII inhibition in a mouse model of venous thrombosis

Introduction

Partial inhibition of Factor VIII (FVIII) may provide antithrombotic efficacy whilst avoiding excessive anticoagulation.

Materials and Methods

We studied the anticoagulant effects of a partial (TB-402) and a complete (BO2C11) FVIII-inhibiting monoclonal antibody (MAb) on FVIII, aPTT, thrombin generation and fibrin deposition in a flow chamber model. The antithrombotic efficacy of TB-402 and BO2C11 was compared in a mouse model of venous thrombosis.

Results

Both in vitro and ex vivo, the maximally achievable FVIII inhibition by TB-402 was about 25 to 30%. The degree of inhibition reached a plateau in vitro at 0.316 μg/mL and ex vivo after administering 0.1 mg/kg and higher doses. BO2C11 strongly inhibited FVIII:C, up to 91% at 100 μg/mL in vitro, and by 88% ex vivo 1 hour after administering 1 mg/kg to the mice.Whereas BO2C11 also markedly prolonged the aPTT and completely inhibited thrombin generation in vitro and ex vivo, the effect of TB-402 on the aPTT and on thrombin generation was limited. Similarly, in a dynamic flow chamber model, TB-402 and BO2C11 inhibited tissue factor-induced human fibrin deposition by 40% and 76%, respectively.In a mouse model of FeCl₃-induced venous thrombosis, TB-402 (1 mg/kg) inhibited thrombus formation to the same extent as BO2C11 (2 mg/kg) and enoxaparin (5 mg/kg), with a mean (± SD) occlusion time of 51 ± 13 minutes for TB-402, compared to 28 ± 6 minutes for the controls, 51 ± 13 minutes for BO2C11 and 55 ± 11 minutes for enoxaparin.

Conclusions

In this mouse model of venular thrombosis, partial FVIII inhibition yielded similar antithrombotic effects as nearly complete FVIII inhibition. These preclinical data are indicative of a therapeutic potential of partial FVIII inhibition in the management of venous thromboembolism.     

Changes in plasma von Willebrand factor and ADAMTS13 levels associated with left atrial remodeling in atrial fibrillation

Introduction

Previous studies have shown raised plasma von Willebrand factor (VWF) levels in patients with atrial fibrillation (AF). However, little is known about changes of VWF associated with VWF-cleaving protease (ADAMTS13) in AF. The aim of this study was to examine the relationship between changes in plasma VWF and ADAMTS13 levels, and left atrial remodeling in AF patients.

Materials and Methods

We measured plasma VWF and ADAMTS13 antigen levels in 70 paroxysmal AF (PAF) patients, 56 chronic AF (CAF) patients, and 55 control subjects.

Results

Plasma VWF levels (mU/ml) were significantly higher in CAF and PAF patients compared with the controls (2103 ± 743, 1930 ± 676, 1532 ± 555, respectively, P < 0.0001 in CAF vs. controls, P = 0.001 in PAF vs. control), while ADAMTS13 levels (mU/ml) were significantly lower in CAF and PAF patients compared with the controls (795 ± 169, 860 ± 221, 932 ± 173, respectively, P = 0.0002 in CAF vs. controls, P = 0.04 in PAF vs. control). The VWF/ADAMTS13 ratio was significantly higher in patients with CAF than PAF or controls (2.81 ± 1.30, 2.34 ± 0.92, 1.73 ± 0.83, respectively; P = 0.01 in CAF vs. PAF, P < 0.0001 in CAF vs. controls). There was a significant correlation between the VWF/ADAMTS13 ratio and left atrial diameter (positive correlation; r = 0.275, P = 0.0002) and left atrial appendage flow velocity (negative correlation; r = - 0.345, P = 0.0018).

Conclusions

These findings suggest that the imbalance between plasma VWF and ADAMTS13 levels caused by left atrial remodeling might be closely associated with intra-atrial thrombus formation in AF patients.     

Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model

Introduction

The prevalence of cardiovascular diseases, one of the major causes of worldwide mortality, is being increasingly reported. Safer, more effective, and less expensive thrombolytic drugs can possibly overcome the underlying problems associate with current thrombolytic drugs.

Methods

A thrombolytic enzyme was purified and characterized from a Streptomyces strain. Carrageenan induced tail-thrombosis mice model was used to evaluate in vivo antithrombotic effect of the enzyme.

Results

First 15 N-terminal amino acids of the purified enzyme were IAGGQAIYAGGGRRS, which are significantly different from the reported fibrinolytic enzymes. The enzyme exhibited 14.3 ± 2.3-fold stronger thrombolytic activity than that of plasmin. In carrageenan induced tail-thrombosis model, the enzyme caused reduction in frequency of thrombus. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose.

Conclusions

The enzyme purified from the Streptomyces strain can be a potential candidate for the treatment of thrombosis.     

Influence of GGCX genotype on warfarin dose requirements in Chinese patients

Introduction

It has been widely accepted that genetic factors were the major sources of the variation in warfarin dose. This study is intended to investigate whether the 3261G>A variation in GGCX gene influences stable warfarin dose in Chinese patient population.

Materials and Methods

A total of 217 patients with stable warfarin dose were enrolled. Genomic DNA was extracted from each subject and the genotype of GGCX 3261G>A was determined by using of denaturing high-performance liquid chromatography (DHPLC). Least significant difference tests (LSDs) were used to compare dose with genotypes. Analysis of variance (ANVOA) was used to calculate the proportion of warfarin dose that could be explained by variation in genotype.

Results

In the total of 217 subjects, 84 patients (38.7%) were GG homozygote, whereas 117 (53.9%) were GA heterozygote and 16 (7.4%) were AA homozygote. Patients with the GGCX 3261AA genotype had a significantly higher average daily maintenance dose (3.39 ± 1.40 mg) than those with the GG genotype (2.69 ± 1.07 mg; P = 0.027), and GGCX 3261G>A explains 2.3% of the univariate warfarin dose variance.

Conclusion

GGCX 3261G>A may affect warfarin dose requirements, and showed a small but significant effect on warfarin dose in a Chinese patient population.     

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.     

Haemostatic effects of phytoestrogen genistein in postmenopausal women

Introduction

Genistein is an isoflavone phytoestrogen derived from the soybean which acts as natural selective estrogen receptor modulator. Various studies have pointed out its cardioprotective role. The aim of the study was to evaluate the haemostatic effects of genistein in postmenopausal women.

Material and methods

In this double-blind placebo-controlled trial we enrolled 104 healthy postmenopausal women with osteopenia. 53 patients (mean age 54.9 ± 4.2 yr; BMI 23.4 ± 3.2 Kg/m²) received genistein (54 mg/day) and 51 patients (mean age 55.4 ± 4.3 yr; BMI 23.6 ± 3.6 Kg/m²) received an identical placebo-tablet. Both groups received a calcium and vitamin D supplement. Plasma levels of D-dimer (DD), plasminogen activator inhibitor-1 (PAI-1) and prothrombin fragment 1 + 2 (F1 + 2) were measured at baseline and after 6 and 12 months of treatment.

Results

Baseline characteristics of the two groups were similar. Compared with placebo, genistein decreased significantly DD (p < 0.001), but did not affect PAI-1 and F 1 + 2 plasma levels.

Conclusion

The results of our study do not confirm effects of genistein on activation of the haemostatic system, but on the contrary the significant decrease of DD could indicate a possible cardioprotective role of genistein in postmenopausal women.     

In vitro comparison of dabigatran, unfractionated heparin, and low-molecular-weight heparin in preventing thrombus formation on mechanical heart valves

Introduction

Lifelong oral anticoagulation (OAC) therapy is required for the prevention of thromboembolic events after implantation of an artificial heart valve. Thromboembolism and anticoagulant-related bleedings account for ≈ 75% of all complications experienced by heart valve recipients (2-9% of patients per year). The present study investigated the efficacy of dabigatran, a new direct thrombin inhibitor for oral use, as compared to unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in preventing thrombus formation on mechanical heart valves in vitro.

Material and Methods

Blood (230 ml) from healthy young male volunteers was anticoagulated either by dabigatran (1 μmol/l), UFH (150 IU), or LMWH (100 IU). Mechanical heart valve prostheses were placed in an in vitro thrombosis tester and exposed to the anticoagulated blood samples under continuous circulation at a rate of 75 beats per minute.

Results

In whole blood with no anticoagulant, the apparatus completely clotted in 15-20 minutes. When blood was treated with dabigatran, the mean thrombus weight was 164 ± 55 mg, in the UFH group 159 ± 69 mg, and in the LMWH group 182 ± 82 mg (p-value: 0.704). Electron microscopy showed no significant difference in thrombus formation in any group.

Conclusisons

Dabigatran was as effective as UFH and LMWH in preventing thrombus formation on mechanical heart valves in our in vitro investigation. Thus, we hypothesize that dabigatran etexilate might potentially be a useful and competitive orally administered alternative to UFH and LMWH for recipients of alloplastic heart valve prostheses.     

Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism

Background

Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Microparticles (MPs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play a role in VTE, although evidence of this association is still limited.

Objectives

To evaluate the hypothesis that elevated numbers of endothelial (EMPs), platelets (PMPs), and Tissue Factor-bearing MPs (TF⁺MPs) in plasma may contribute to cancer-associated thrombosis.

Patients/Methods

EMPs, PMPs and TF⁺MPs plasma levels were measured in 90 consecutive patients (cases) referred to our Department (30 with a first episode of unprovoked VTE; 30 with active cancer; 30 with a diagnosis of acute VTE associated with active cancer), and in a group of 90 healthy subjects (controls). MPs analyses were performed by flow-cytometry (Cytomics FC500).

Results

Cases showed statistically significant higher (mean ± SD) circulating EMPs and PMPs plasma levels (920 ± 341 and 1221 ± 413 MP/μL, respectively) than controls (299 ± 102 and 495 ± 241 MP/μL; p < 0.005). Moreover cancer patients (with and without VTE) showed higher (mean ± SD) TF⁺MPs (927 ± 415 MPs/μL) than controls (204 ± 112 MPs/μL; p < 0.001). The subgroup of cancer patients plus VTE showed statistically significant higher TF⁺MPs plasma levels (1019 ± 656 MPs/μL) than cancer patients without VTE (755 ± 391 MPs/μL, p = 0.002). Multivariate analysis failed to show a significant association between elevated TF⁺MPs and VTE in cancer patients.

Conclusions

Our results suggest that MPs might be an important intermediate in the cascade of cellular injury and vascular dysfunctions underlying the process of thrombosis, particularly in cancer. Further clinical investigations are needed to confirm the precise role of MPs in predicting hypercoagulable state in patients with cancer.     

Effects of risperidone on glucose metabolism in Chinese patients with schizophrenia: a prospective study

Background

While most of the second generation antipsychotic agents are associated with abnormal glucose metabolism, previous studies have shown that risperidone has relatively little effect upon blood glucose levels. This study aimed to explore the effect of risperidone on the glucose-regulating mechanism of patients with schizophrenia by using the oral glucose tolerance test (OGTT), measuring insulin and C-peptide levels.

Methods

Thirty inpatients with schizophrenia taking risperidone were studied. All the patients were given a simplified OGTT at baseline and six weeks after treatment. Plasma glucose, insulin, and C-peptide concentrations were measured at fasting, then 1 and 2 h after OGTT respectively. Other data, including demographic characteristics and plasma drug concentrations, were also recorded.

Results

(1) There was no significant increase in the proportion of patients demonstrating abnormal plasma glucose levels compared with baseline (p = 1.000, McNemar test); (2) risperidone was associated with elevated insulin concentrations (p = 0.013), C-peptide levels (p = 0.020), insulin/glucose ratio (p = 0.020) and BMI (p < 0.01); (3) no sex differences in glucose-related measures were observed.

Conclusion

Risperidone treatment may be associated with alterations in glucose-regulating mechanisms in patients with schizophrenia.     

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.     

A case-control study provides evidence of association for a common SNP rs974819 in PDGFD to coronary heart disease and suggests a sex-dependent effect

Introduction

Peden et al. have revealed a significant association between four new risk loci and coronary heart disease (CHD) in Europeans and South Asians. The goal of this study is to evaluate the contribution of these genetic loci to CHD risk in Han Chinese.

Methods

We recruited 161 CHD patients and 112 controls proved by angiography originated from Ningbo in the Eastern China, and performed a case-control association study of the four significant SNPs.

Results

Among the four tested SNPs, we found a significant association of rs974819 in PDGFD gene with CHD (allele p = 0.04; OR = 1.45, 95% CI = 1.02 - 2.08) and the allele A/G of rs974819 shows significant difference in females (allele p = 0.04; OR = 1.83, 95% CI = 1.01 - 3.31). A further meta-analysis showed that rs974819 of PDGFD gene was significantly associated with an increasing risk of CHD (OR = 1.08, 95% CI = 1.05 - 1.11) in both Europeans and South Asians including Han Chinese.

Conclusions

Our findings suggests that rs974819 of PDGFD is also a CHD risk factor in Han Chinese. In addition, it presents a sex-dependent genetic effect.     

Platelet activation in patients with peripheral vascular disease: reproducibility and comparability of platelet markers

Background

Many markers of platelet activation have been described but their reproducibility and comparability in patient populations are poorly defined.

Objectives

We sought to compare markers of platelet and monocyte activation with platelet-monocyte aggregates, a proposed gold standard of in vivo platelet activation, and assess their reproducibility in patients with peripheral arterial disease: a population with substantial platelet activation, inflammation and risk of thrombotic events.

Patients/Methods

Thirty patients with peripheral vascular disease attended on two occasions to permit within-day and between-day comparisons. In vivo platelet and monocyte activation were determined by flow-cytometric quantification of platelet-monocyte aggregation, platelet surface expression of P-selectin and CD40L, platelet-derived microparticles, and monocyte surface expression of CD40 and CD11b. Plasma concentrations of platelet-derived microparticles, soluble P-selectin and CD40L were measured by enzyme-linked immunosorbant assays.

Results

Platelet-monocyte aggregation (36.7 ± 7.86%), and platelet surface expression of P-selectin (5.8 ± 1.65%) and CD40L (3.3 ± 1.45%) demonstrated comparable within-day (mean difference ± co-efficient of reproducibility; 0.9 ± 15.4%, 0.21 ± 1.65% and 0.2 ± 2.8% respectively) and between-day reproducibility (2.0 ± 12.4%, 0.10 ± 2.25% and 0.9 ± 6.4% respectively). Platelet-monocyte aggregates correlated well with other platelet (r = 0.30-0.50, P < 0.02) and monocyte (r = 0.27-0.47, P < 0.03) activation markers. Flow cytometric and assay quantified platelet-derived microparticles showed poorer reproducibility (co-efficient of reproducibility > 40).

Conclusions

In patients with peripheral arterial disease, measurements of platelet-monocyte aggregates have good reproducibility and consistently reflect other markers of platelet and monocyte activation.     

Automated mixing studies and pattern recognition for the laboratory diagnosis of a prolonged activated partial thromboplastin time using an automated coagulation analyzer

Introduction

The objective of this study was to explore whether an automated coagulation analyzer could be applied to normal plasma mixing studies for the assessment of blood samples showing a prolonged activated partial thromboplastin time (APTT).

Materials and methods

Ten laboratory staff members performed normal plasma mixing studies and evaluated plasma samples using 3 different methods: (1) manual dilution and analysis, (2) manual dilution and automatic analysis with STA-R^®, and (3) automatic dilution and analysis with the Coapresta^® 2000 (CP2000). The time from the start of the analysis to the generation of the result plots and the plasma volumes required were determined. We analyzed patient plasma samples showing a prolonged APTT using the CP2000, and the result plots were categorized into 3 curve patterns based on the area ratio values: the inhibitor type (convex pattern), deficiency type (concave pattern), and suspicious inhibitor type (approximately straight pattern).

Results

When pooled patient plasma was used, the same patterns were obtained from normal plasma mixing studies using the 3 different methods. The time required to complete the mixing studies and the plasma volumes required were 28.2 ± 2.4 min and 350 μL for manual analysis, 23.2 ± 2.1 min and 875 μL for STA-R^®, and 8.5 ± 0.1 min and 175 μL for CP2000, respectively. Of 31 patient samples, 9 were categorized into the inhibitor type, 15 were categorized into the deficiency type, and 7 were categorized into the suspicious inhibitor type.

Conclusions

The CP2000 analyzer is applicable to the laboratory diagnosis of a prolonged APTT using pattern recognition, as it requires a shorter time to complete mixing studies and a smaller plasma volume in comparison with manual analysis.     

The impact of selective and non-selective non-steroid anti-inflammatory drugs on secondary hemostasis in healthy volunteers

Background

Clinical and epidemiological studies have associated selective COX-2 inhibitors with an increased risk of cardiovascular events. There are no clinical studies on the possible effects of these drugs on secondary hemostasis. The hypothesis for this study is that the use of selective COX-2 inhibitors could affect the secondary hemostasis and by that increase the risk of cardiovascular events in a population at high risk.

Methods

An open-label randomized cross-over study was performed in 20 healthy male volunteers. The study consisted of two periods of each 21 days with medication, either celecoxib 100 mg b.i.d. or naproxen 250 mg b.i.d. Treatment periods were separated by a washout period of 28 days. Blood samples were obtained before the first medication period, and at the end of each medication period. Primary effect parameter was FXII level. Secondary effect parameters included a wide range of coagulation factors involved in secondary hemostasis.

Results

There was no statistically significant effect of celecoxib or naproxen on the primary effect parameter. Protein C activity was significantly decreased after treatment with naproxen (P < 0.01), compared to baseline. Platelet function, demonstrated as closure time (CT), was at baseline 118 ± 24 sec. (mean ± SD). Naproxen prolonged CT to 171 ± 50 sec. (P < 0.001). Celecoxib did not change CT significantly (119 ± 24 sec.).

Conclusions

Neither the selective COX-2 inhibitor celecoxib, nor the non-selective NSAID naproxen caused any change in the primary effect parameter from the secondary hemostasis.     

Antithrombin and heparin cofactor II-mediated inactivation of α-thrombin by a synthetic, sulfated mannogalactan

Introduction

A mannogalactan from Pleurotus ostreatoroseus (MgPr) was chemically sulfated to give MgPr-S1, which was evaluated for its anticoagulant and antithrombotic activities, bleeding tendency, and platelet aggregation.

Materials and methods

MgPr-S1 was partially characterized by HPSEC-MALLS, methylation analysis, and ¹³C NMR spectroscopy. Its anticoagulant activity was determined by assays of aPTT, TT, α-thrombin and factor Xa residual activity, heparin cofactor II (HCII)-, or antithrombin (AT)-mediated inhibition. The antithrombotic effect was evaluated in rats using a venous thrombosis model and the bleeding tendency was also tested in vivo. Platelet aggregation was investigated by an adaptation of the method of Born [1].

Results

The hydroxyl groups of β-D-Manp units and OH-2 and OH-4 of the (1→6)-linked α-D-Galp units were preferentially substituted. The anticoagulant activity of MgPr-S1 was mainly by thrombin inhibition with antithrombin and HCII, and had an effect on platelet aggregation induced by ADP and α-thrombin. It almost completely inhibited thrombus formation in vivo at a dose of 6 mg/kg and heparin inhibited thrombus formation at a dose of 0.200 mg/kg.

Conclusions

These results suggested that the chemically sulfated mannogalactan could act as an alternative to heparin as anticoagulant.     

Aspirin insensitive thromboxane generation is associated with oxidative stress in type 2 diabetes mellitus

Introduction

Aspirin (ASA) irreversibly inhibits platelet cyclooxygenase-1 (COX-1) leading to decreased thromboxane-mediated platelet activation. The effect of ASA ingestion on platelet activation, thromboxane generation, oxidative stress and anti-oxidant biomarkers was studied in type 2 diabetes mellitus (DM).

Material and methods

Baseline and post-ASA samples (100/325 mg x 7 days) were obtained from 75 DM patients and 86 healthy controls for urinary 11-dehydro-thromboxane B2 (11dhTxB2), 8-iso-prostaglandin-F2α (8-isoPGF2α) and serum sP-Selectin, nitrite (NO₂^-), nitrate (NO₃^-) and paraoxonase 1 (PON1) activity.

Results

Compared to baseline controls, baseline DM had higher mean levels of 11dhTxB2 (3,665 ± 2,465 vs 2,450 ± 1,572 pg/mg creatinine, p = 0.002), 8-isoPGF2α (1,457 ± 543 vs 1,009 ± 412 pg/mg creatinine, p < 0.0001), NO₂^- (11.8 ± 7.3 vs 4.8 ± 5.3 μM, p < 0.0001), NO₃^- (50.4 ± 39.3 vs 20.9 ± 16.7 μM, p < 0.0001) and sP-Selectin (120.8 ± 56.7 vs 93.0 ± 26.1 ng/mL, p = 0.02), and the same held for post-ASA levels (p < 0.0001). ASA demonstrated no effect on 8-isoPGF2α, NO₂^-, NO₃^-, sP-Selectin or PON1 activity in either DM or controls. Post ASA inhibition of urinary 11dhTxB2 was 71.5% in DM and 75.1% in controls. There were twice as many ASA poor responders in DM than in controls (14.8% and 8.4%) based on systemic thromboxane reduction. Urinary 8-isoPGF2α excretion was greater in DM ASA poor responders than good responders (p < 0.009).

Conclusions

This suggests that oxidative stress may maintain platelet function irrespective of COX-1 pathway inhibition and/or increase systemic generation of thromboxane from non-platelet sources.