Platelet activation and endogenous thrombin potential in pre-eclampsia

Introduction

Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia.

Materials/methods

Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n = 46) and without (n = 46) pre-eclampsia, and in healthy non-pregnant women (n = 42).

Results

The percentage of, CD62P+ platelets (p = 0.013), CD62P+ platelet microparticles (p = 0.029) and platelet-monocyte aggregates (p = 0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p  < 0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p < 0.001) than the normotensive pregnant controls.

Conclusion

In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.     

Endogenous thrombin potential (ETP) in plasma from patients with AMI during antithrombotic treatment

Background

The beneficial impact of warfarin in preventing new events after AMI is well established. Decrease in thrombin generation seems to be the key element in anticoagulant treatment.

Objectives

The aims were to investigate the effect of warfarin and platelet inhibition on thrombin generation, assessed by the endogenous thrombin potential (ETP), and study the relation between coagulation parameters and ETP in patients with AMI.

Patients/Methods

In the present sub-study of the WARIS II trial, patients with AMI were randomly assigned to treatment with aspirin 160 mg/d (n = 57), aspirin 75 mg/d and warfarin (INR 2.0-2.5) (n = 68) or warfarin (INR 2.8-4.2) (n = 61). Fasting blood samples were collected from patients at discharge from hospital and after 6 weeks treatment.

Results

Correlation analyses showed that both ETP and peak thrombin levels were significantly correlated with Factor VII Ag (r = 0.38 and 0.36 respectively, p < 0.01 for both) and with F1+2 (r = 0.26 and 0.23 respectively, p = 0.01 for both) at baseline. Antithrombotic treatment for 6 weeks caused a highly significant inhibition of ETP in patients treated with warfarin (− 28% ± 5%, p < 0.001), and patients treated with aspirin/warfarin (− 24% ± 8%, p = 0.04). Similarly, peak thrombin levels were reduced in patients treated with warfarin (− 18% ± 7%, p = 0.049) and aspirin/warfarin (− 19% ± 5%, p = 0.029), whereas an increase (12% ± 4%, p = 0.029) occurred during aspirin treatment alone. F1+2 levels decreased by 64% and 58% in the warfarin and aspirin/warfarin groups, respectively (p = 0.001 for both).

Conclusions

In patients with AMI, warfarin significantly reduced the endogenous thrombin generation and the potential to generate thrombin in plasma ex vivo, whereas aspirin alone had no effect on thrombin generation in vivo or ex vivo, assessed by ETP.     

Reference intervals for platelet aggregation assessed by multiple electrode platelet aggregometry

Introduction

Analyses of platelet aggregation in hirudin whole blood using Multiplate® was validated. Reference intervals for the most commonly used agonists were established, and the association between platelet aggregation, age, gender and haematological values was analysed.

Material and methods

We included 121 healthy individuals to establish reference intervals and six healthy individuals for evaluation of the day-to-day variation. Platelet aggregation was evaluated on hirudin whole blood employing Multiplate® induced by arachidonic acid, ADP, collagen and ristocetin (RISTOlow and RISTOhigh). Measurements of haematological values were performed employing Sysmex K-4500.

Results

We found no association between platelet aggregation and age (p > 0.57 for all agonists, except RISTOlow: p = 0.05). Platelet aggregation was significantly higher in women compared to men for all agonists (p < 0.0003), except RISTOlow (p = 0.05). A reference interval is presented as 95% confidence interval suitable for any age and both sex. Day-to-day variation was < 11% for all agonists except for RISTOlow. No association was found between platelet aggregation and haematocrit or red blood cell count after adjusting for age and gender except for RISTOhigh. A positive significant association was found between platelet count and platelet aggregation (p < 0.04). Finally, a significant positive association was found between platelet aggregation and white blood cell count for all agonists (p < 0.05) except RISTOlow and RISTOhigh (p > 0.05).

Conclusion

Reference intervals for platelet aggregation in healthy individuals (age: 17 to 66 years) were established in hirudin whole blood measured by Multiplate® employing the most commonly used agonists.     

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

Background

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

Methods

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

Results

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

Conclusions

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

The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression

Introduction

High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs).

Material and Methods

DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI.

Results

In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p = 0.02) and to granulocytes (p = 0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p = 0.02-0.002). In the MI population, the DTI reduced the TF activity (p < 0.001).

Conclusion

DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production.     

Influence of renal function and platelet turnover on the antiplatelet effect of aspirin

Introduction

Kidney disease predisposes to cardiovascular events. This study investigated the influence of renal function and platelet turnover on the antiplatelet effect of aspirin in patients with coronary artery disease.

Materials and Methods

We included 124 aspirin-treated patients with coronary artery disease and normal to moderately reduced renal function. All tests were performed one hour after aspirin ingestion. Renal function was assessed using creatinine, estimated glomerular filtration rate (eGFR), and cystatin C. The antiplatelet effect of aspirin was evaluated using the VerifyNow® Aspirin assay and multiple electrode aggregometry (MEA, Multiplate®) induced by collagen (1.0 μg/mL) and arachidonic acid (1.0 mmol/L). Von Willebrand factor was measured as a marker of endothelial dysfunction. Platelet turnover was evaluated by measurements of immature, reticulated platelets.

Results

Renal function did not influence the antiplatelet effect of aspirin evaluated by MEA (r = − 0.2-0.09, p = 0.03-0.77) or the VerifyNow® (r = − 0.12-0.11, all p-values > 0.1). In contrast, renal function correlated inversely with von Willebrand factor levels (r_creatinine = 0.48, p < 0.0001; r_eGFR = − 0.46, p < 0.001; r_{cystatin C} = 0.54, p < 0.0001). The number of immature platelets correlated with platelet aggregation according to MEA (r = 0.20-0.39, all p-values < 0.03), but not according to VerifyNow® (r = − 0.07, p = 0.50).

Conclusions

A reduced antiplatelet effect of aspirin may be explained by an increased number of immature platelets. Moderately impaired renal function was associated with high levels of von Willebrand factor, but not with a reduced antiplatelet effect of aspirin.     

Evaluation of platelet function and pharmacological platelet inhibition in patients with myeloproliferative disorders using multiple electrode aggregometry

Background

The aim of this study was to describe platelet aggregation characteristics by multiple electrode aggregometry (MEA) and to evaluate MEA for its potential to detect platelet dysfunction and response to anti-aggregatory drugs in patients with myeloproliferative disorders (MPD).

Methods

We compared the platelet response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) in hirudin-anticoagulated blood of 55 patients with polycythaemia vera and essential thrombocythaemia and 75 controls.

Results

Comparing MPD patients and controls no statistically significant difference indicative of platelet dysfunction was found in MPD patients. Analysis of covariance revealed platelet- and leukocyte count as a significant influencing factor on MEA function. Furthermore we could demonstrate that ASA and clopidogrel treatment results in a statistically significant lower ASPI (Controls: p < 0.0001, MPD: p < 0.0001) and ADPtest value (MPD: p = 0.00125) compared to untreated patients thereby validating the method for monitoring of anti-aggregatory therapy.

Conclusion

In this study MEA was confirmed as a valid method for monitoring of ASA and clopidogrel treatment in patients with MPD and normal control subjects. The platelet and leukocyte count were identified as major influencing factors on MEA aggregation tests both in MPD patients and controls. No functional platelet abnormalities were detected in MPD patients.     

Continuous thrombin infusion leads to a bleeding phenotype in sheep

Background

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo.

Methods

Thrombin (0.0004-0.42 IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n = 5 animals; 15 experiments). In the dose verification study animals received 0.42 IU/kg/min of thrombin vs. saline solution in a cross-over design (n = 3 animals; 7 experiments).

Results

Thrombin at an infusion rate of 0.42 IU/kg/min decreased fibrinogen levels by 75% (p < 0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p = 0.006), prolonged thrombin time by 70% (p = 0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p < 0.05) and decreased the activity of the coagulation factors VIII and XIII (p < 0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p = 0.019) and induced a 65% decrease in maximal clot firmness (p < 0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism.

Conclusion

Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.     

Platelet aggregation is dependent on platelet count in patients with coronary artery disease

Introduction

Platelet function testing in whole blood is widely used to evaluate the effect of antiplatelet agents, but it is not known whether results are affected by whole blood parameters. This study investigated the importance of platelet count, haematocrit, red blood cells (RBC), and white blood cells in whole blood platelet aggregometry.

Materials and methods

We included 417 patients with coronary artery disease on aspirin mono-therapy and 21 aspirin-naïve healthy individuals. Blood sampling was performed one hour after aspirin ingestion. The antiplatelet effect of aspirin was evaluated using the VerifyNow® Aspirin assay and multiple electrode aggregometry (MEA, Multiplate®) induced by collagen (1.0 μg/mL) and arachidonic acid (1.0 or 0.75 mmol/L). Measurements of whole blood parameters were performed to evaluate the three major cell lines in circulating blood.

Results

In patients, platelet count correlated significantly with platelet aggregation (MEA_collagen, p < 0.0001; MEA_{arachidonic acid}, p < 0.0001; VerifyNow®, p = 0.03). Haematocrit and RBC correlated inversely with MEA induced by collagen (p_haematocrit < 0.001; p_RBC = 0.07) and with VerifyNow® (p_haematocrit < 0.0001; p_RBC < 0.0001), but not with MEA induced by arachidonic acid (p_haematocrit = 1; p_RBC = 0.87). White blood cells correlated significantly with platelet aggregation (MEA_collagen, p < 0.001; MEA_{arachidonic acid}, p < 0.0001; VerifyNow®, p = 0.05). Similar associations were observed in aspirin-naïve healthy individuals.

Conclusions

Whole blood aggregometry is dependent on all major cell lines in whole blood. Importantly, platelet aggregation is significantly associated with platelet count even within the normal range.     

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Background

The calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma.

Objective

Use the CAT assay to detect endogenous tissue factor (TF) in recalcified platelet-rich plasma (PRP) and platelet-free plasma (PFP).

Methods

Blood from healthy volunteers was collected into citrate and incubated at 37 °C with or without lipopolysaccharide (LPS) for 5 hours. PRP and PFP were prepared and clotting was initiated by recalcification. Thrombin generation was measured using the CAT assay.

Results

The lag time (LT) was significantly shortened in PRP prepared from LPS-treated blood compared with untreated blood (10 ± 3 min versus 20 ± 6 min), and this change was reversed by the addition of inactivated human factor VIIa. LPS stimulation did not change the peak thrombin. Similar results were observed in PFP (21 ± 4 min versus 35 ± 5 min). LPS stimulation also significantly reduced the LT of PRP and PFP derived from blood containing citrate and a factor XIIa inhibitor. Finally, a low concentration of exogenous TF shortened the LT of PFP prepared from unstimulated, citrated blood without affecting the peak thrombin.

Conclusion

Changes in LT in the CAT assay can be used to monitor levels of endogenous TF in citrated plasma.     

Protease activated receptor-1 (PAR-1) mediated platelet aggregation is dependent on clopidogrel response

Introduction

Clopidogrel inhibits ADP mediated platelet aggregation through inhibition of the P2Y12 receptor by its active metabolite. Thrombin induces platelet aggregation by binding to protease activated receptor-1 (PAR-1), and inhibition of PAR-1 has been evaluated in patients treated with clopidogrel to reduce ischemic events after acute coronary syndromes. Residual PAR-1 mediated platelet aggregation may be dependent on extent of clopidogrel response.

Material and Methods

Platelet aggregation was measured in 55 patients undergoing elective PCI at 16-24 hours after 600 mg clopidogrel loading dose by light transmittance aggregometry using ADP 20 μM and thrombin receptor agonist peptide (TRAP) at 15 μM and 25 μM as agonists. Genomic DNA was genotyped for common CYP2C19 variants.

Results

Increasing quartiles of 20 μM ADP induced platelet aggregation after clopidogrel loading were associated with increasing levels of TRAP mediated platelet aggregation. Patients in the highest quartile (clopidogrel non-responders) of post treatment ADP aggregation had significantly higher TRAP mediated aggregation than the patients in the lowest quartile (clopidogrel responders) [TRAP 15 μM: 79.6 ± 5% vs. 69.5 ± 8%, p < 0.001].

Conclusions

Non-responders to clopidogrel show increased residual platelet aggregation induced by TRAP, whereas clopidogrel responders exhibit attenuated response to TRAP. Addition of PAR-1 antiplatelet drugs may be most effective in patients with reduced clopidogrel response and high residual TRAP mediated platelet aggregation.     

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction

Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition.

Methods

Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients.

Results

In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r² = 0.33 and r² = 0.34 respectively, p < 0.0015). In patients with a low platelet count (< 150 × 10⁹ platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r² = 0.33 and r² = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05).

Conclusions

There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count.     

Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma

Introduction

In an effort to administer life-saving transfusions quickly, some trauma centers maintain thawed plasma (TP). According to AABB, TP is approved for transfusion for up to five days when stored at 1 - 6 °C. However, the alterations in microparticles (MP) contained in the plasma, which are an integral component of plasma's hemostatic capacity, are not well characterized. We report on MP changes in TP between its initial thaw (FFP-0) and five days (FFP-5) of storage.

Materials and Methods

FFP units (n = 30) were thawed at 37 °C and kept refrigerated for five days. Phenotypes of residual cells, which include platelets, erythrocytes, leukocytes, monocytes, endothelial cells, and MP counterparts of each cell type, were analyzed by flow cytometry. Functional assays were used for MP procoagulant activity, plasma thrombin generation, and clotting properties (thromboelastography).

Results

In FFP-0 the majority (94%) of residual cells were platelets, along with significant levels of platelet MPs (4408 × 10³/L). FFP-5 showed a decline in MP count by 50% (p < 0.0001), and procoagulant activity by 29% (p < 0.0001). FFP-5 exhibited only 54% (p < 0.0001) of the potential for thrombin generation as FFP-0, while thromboelastography indicated a slower clotting response (p < 0.0001) and a longer delay in reaching maximum clot (p < 0.01). Removal of MP by filtration resulted in reduced thrombin generation, while the MP replacement restored it.

Conclusions

Decline in MP with storage contributes to FFP-5's reduced ability to provide the hemostatic potential exhibited by FFP-0, suggesting the presence of platelet MPs in freshly TP may be beneficial and protective in the initial treatment of hemorrhage.     

Is switching antidepressants following early nonresponse more beneficial in acute-phase treatment of depression?: a randomized open-label trial

Rationale

Treatment guidelines for major depressive disorder (MDD) recommend a continuous use of antidepressants for several weeks, while recent meta-analyses indicate that antidepressant efficacy starts to appear within 2 weeks and early treatment nonresponse is a predictor of subsequent nonresponse.

Objectives

We prospectively compared 8-week outcomes between switching antidepressants and maintaining the same antidepressant in early nonresponders, to generate a hypothesis on possible benefits of early switching strategy.

Method

Patients with MDD without any treatment history for the current episode were included. When subjects failed to show an early response (i.e., ≥ 20% improvement in the Montgomery-Åsberg Depression Rating Scale (MADRS)) to the initial treatment with sertraline 50 mg at week 2, they were randomly divided into two groups; in the Continuing group, sertraline was titrated at 50-100 mg, whereas sertraline was switched to paroxetine 20-40 mg in the Switching group. A primary outcome measure was a response rate (i.e., ≥ 50% improvement in the MADRS) at week 8.

Results

Among 132 subjects, 41 subjects showed early nonresponse. The Switching group (n = 20) showed a higher rate of responders than the Continuing group (n = 21) (75% vs. 19%: p = 0.002). Further, the Switching group was also superior in the rate of remitters (total score of ≤ 10 in the MADRS) (60% vs. 14%: p = 0.004) and continuous changes in the MADRS (19.0 vs. 7.5: p < 0.001).

Conclusions

Our preliminary findings suggest that patients with MDD who fail to show early response to an initial antidepressant may derive benefits from the early switching antidepressants in the acute-phase treatment of depression.     

Lower tissue factor inhibition in patients with ST segment elevation than in patients with non ST elevation acute myocardial infarction

Introduction

Mechanisms to explain the different course of coronary thrombosis between ST elevation myocardial infarction (STEMI) and non-STEMI patients remain poorly defined. We hypothesize, however, that STEMI patients may present lower tissue factor plasma inhibition to partly account for their more persistent coronary thrombotic occlusion.

Materials and Methods

Total (t-TFPI ) and free tissue factor plasma inhibitor (f-TFPI), thrombin-antithrombin complex (TAT), plasminogen activator inhibitor 1 (PAI-1), von Willebrand factor (vWF), and fibrinogen were measured on admission and at 3 and 6 months in patients with a first STEMI (n:69) or non-STEMI (n:60). C reactive protein (CRP) was also measured on admission and at 3 months.

Results

STEMI patients showed lower admission levels of t-TFPI (p = 0.001), f-TFPI (p = 0.030) and fibrinogen (p = 0.022), and higher vWF levels (p = 0.005) than non-STEMI whereas TAT, PAI and CRP levels were comparable. At 3 and 6 months VWF, t-TFPI, f-TFPI, and TAT levels declined significantly in the 2 groups (p = 0.002) reaching similar values. CRP levels also declined at 3 months (p = 0.002). Moreover, the rate of cardiac mortality, non fatal MI or stroke during a 6 year follow-up were unrelated to admission coagulation parameters.

Conclusions

The lower inhibition of tissue factor and greater endothelial dysfunction in STEMI than in non-STEMI patients may enhance thrombosis at the culprit lesion and adjacent coronary plaques, and hence, account at least in part for their different pathophysiology. This condition, however, is limited to the acute phase.     

Circulating microparticles are elevated in carriers of Factor V Leiden

Introduction

Microparticles (MP) are small membrane bound cellular particles that play an important role in thrombosis. This study was carried out to evaluate if increased numbers or procoagulant potential of circulating MP contribute to the heterogeneity in occurrence of thrombosis in heterozygotes carrying Factor V Leiden (FVL) mutation.

Methods

Levels of circulating platelet (CD41a), endothelial (CD62e) as well as leukocyte (CD45) derived MP from 45 FVL heterozygous individuals were enumerated by flow cytometry and compared with normal controls. Functional studies included enzyme linked immunoassay based prothrombinase activity (ELISA) and modified dilute Russell Viper venom test (DRVVT).

Results

Circulating MP were significantly higher in the FVL cohort compared to the controls (median = 2100 vs. 1508 MP/μl, respectively p = 0.0021).All subsets of MP (platelet, endothelial and leukocyte) were significantly elevated in the FVL group, the most striking disparity seen in the number of CD45 positive leukocyte MP. Despite the differences in the number of MP between the controls and FVL cohorts, there was no significant difference in the prothrombinase activity recorded by the ELISA (2.0 vs 2.4 PS equivalents; p = 0.7374) or clotting time assessed by the DRVVT (47 vs 46 sec, p = 0.8118). When the FVL cohort was considered alone there was no significant difference in MP parameters between FVL subjects with or without a history of thrombosis.

Conclusions

This is the first study on circulating MP levels in subjects who are heterozygote for factor V Leiden. We report that circulating platelet and leukocyte MP are elevated in carriers of this mutation and may be important contributors to risk of thrombosis.     

Tissue factor-induced Thrombin Generation in the Fasting and Postprandial State among Elderly Survivors of Myocardial Infarction

Introduction

Tissue factor (TF)-induced thrombin generation (TG) ex vivo has been suggested to be an important method to assess thrombotic risk. No studies have investigated the impact of postprandial lipemia on TF-induced TG. Since myocardial infarction (MI) is associated with elevated postprandial levels of triglycerides, we hypothesized a differential impact of postprandial lipemia on coagulation activation in MI-patients and healthy controls.

Material and Methods

Elderly survivors of acute MI (n = 44) and healthy age-and sex matched controls (n = 43) underwent a fat tolerance test (1 gram per kg body weight) to assess coagulation activation during postprandial lipemia.

Results

The incremental area under the curve (AUCi) for serum triglycerides was higher in MI-patients than in healthy age-and sex matched controls (5.64 ± 0.52 mmol/L?h and 3.94 ± 0.39 mmol/L?h, p = 0.012) during the postprandial phase. Subsequent endogenous activation of coagulation, assessed by FVIIa and thrombin generation (F1 + 2), was similar among groups and not related to levels of triglycerides during the postprandial phase. Healthy individuals had a gradual decline in TF-induced thrombin generation ex vivo, assessed by endogenous thrombin potential (ETP) (AUCi = - 542.4 ± 71.4 nM?min?h, p < 0.001), whereas MI-patients retained their ETP (AUCi = 127.4 ± 89.0 nM?min?h, p = 0.47) in plasma during the postprandial phase (p for group difference = 0.005).

Conclusions

MI-patients had elevated postprandial lipemia and retained their ability for TF-induced TG in plasma ex vivo in the postprandial phase, whereas the capacity gradually decreased in healthy individuals. Further studies are warranted to reveal underlying mechanism(s) and clinical implications.     

An experimental model to study isolated effects of thrombin in vivo

Background

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo.

Methods

Thrombin (0.05-0.9 U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n = 38). The study consisted of three parts: dose escalation (n = 21), dose verification (n = 5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n = 12).

Results

A thrombin dose of 0.9 U/kg/min decreased platelet counts by 70% compared to the control group (median 230 × 10^9/L vs. 752 × 10^9/L; p = 0.041). In accordance, infusion of 0.9 U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56 mg/dl vs. 220 mg/dl; p = 0.046). Cumulative thrombin doses of ≥ 0.1 U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥ 0.15 U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9 U/kg/min was associated with a 3 to 6.5 -fold increase in IL-6 levels (139-306 pg/ml vs. 47 pg/ml, p < 0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNF-alpha). In addition, lepirudin prevented thrombin- induced thrombocytopenia.

Conclusion

Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines.     

A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation

Introduction

Z4A5 is a novel peptide that inhibits platelet aggregation and formation of platelet thrombi, but the mechanism of its anti-platelet effects remains unknown. This study explores the anti-platelet effect and mechanism of Z4A5.

Methods

We investigated the anti-platelet activity of Z4A5 on platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin (TH) in human platelet-rich plasma (PRP). Fibrinogen and PAC-1 binding to glycoproteinIIb/IIIa (GPIIb/IIIa) were measured by flow cytometry. In addition, we investigated the integrin specificity of Z4A5 in attachment and detachment assays using human umbilical vein endothelial cells (HUVEC) and assessed the relative cell number using the MTT assay.

Results

In vitro, Z4A5 inhibited ADP-, AA- and TH-induced human platelet aggregation with IC₅₀ values of 0.46 ± 0.05 μM (n = 10), 0.23 ± 0.05 μM (n = 10) and 0.21 ± 0.02 μM (n = 10), respectively. Z4A5 inhibited fibrinogen, and PAC-1 bound to platelet GPIIb/IIIa with IC₅₀ values of 0.48 ± 0.07 μM (n = 8) and 0.63 ± 0.12 μM (n = 6), respectively. Z4A5 failed to inhibit α_Vβ₃ integrin-mediated HUVEC attachment to vitronectin and did not cause any significant detachment of HUVEC monolayer when compared with the controls.

Conclusions

Z4A5 is a potential anti-platelet drug that inhibits fibrinogen binding to GPIIb/IIIa, but does not affect the structurally similar integrin α_Vβ₃.     

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.