Platelet-poor Plasma Samples Research Articles

Effect of Different Storage Conditions on Platelet Aggregation in Horse

Platelet aggregation is the most important event in the hemostatic process, but no information is available on the stability of samples for aggregation testing in horses. The aim of the present study was to evaluate the effect of different storage conditions on platelet aggregation in horses. The study was carried out on 58 healthy horses of varying breed and gender, ranging in age from 4 to 12 years. Citrated blood samples were collected from all the subjects by means of jugular venipuncture and were used for platelet aggregation measurements. Platelet-rich and platelet-poor plasma samples were prepared by centrifugation and divided into six different aliquots to assess the maximum degree of platelet aggregation and the initial velocity of platelet aggregation at the final concentrations of 1 and 0.5 μM of the aggregating agent. The first aliquot was analyzed within 1 hour after collection at room temperature (22°C), the second 6 hours after collection at 22°C, the third and fourth were refrigerated at 8°C for 6 and 24 hours, respectively, and the fifth and sixth were frozen at −20°C for 24 and 48 hours, respectively. With the help of an aggregometer, platelet responses were quantified, and one-way repeated measures analysis of variance was used to determine significant differences. Probability values of <.05 were considered to be statistically significant. Analysis of variance showed statistically significant differences on maximum degree of platelet aggregation and the initial velocity of platelet aggregation using adenosine diphosphate at final concentrations of 1 and 0.5 μM. The results of this study suggest that the storage of equine plasma for more than 6 hours at room temperature and at 8°C has a significant effect on platelet aggregation, and that the storage of plasma for 24 and 48 hours at −20°C alters platelet aggregation. In conclusion, storage conditions had a statistically significant effect on the parameters of platelet aggregation directly correlated to temperature.

Chromatographic analysis of serotonin, 5-hydroxyindolacetic acid and homovanillic acid in dried blood spots and platelet poor and rich plasma samples

An isocratic high-performance liquid chromatographic method has been developed for the measurement of serotonin, 5-hydroxyindolacetic and homovanillic acids in dried blood spots and in platelet poor and rich plasma samples. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 13% methanol and 87% aqueous citrate buffer, containing octanesulfonic and ethylendiaminotetracetic acids. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at −0.200 V and the second analytical cell at +0.400 V. For the pre-treatment of biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange Oasis cartridges, was implemented. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0%. The calibration curve was linear over the on-column concentration range of 0.1–22.5 ng mL −1 for serotonin and 5-hydroxyindolacetic acid and of 0.25–22.5 ng mL −1 for homovanillic acid. The sensitivity was good with a limit of detection of 0.05 ng mL −1 for serotonin and 5-hydroxyindolacetic acid and 0.12 ng mL −1 for homovanillic acid. Results were also satisfactory in terms of precision, selectivity and accuracy. The analytical method was successfully applied to human platelet poor and rich plasma samples and to dried blood spots from volunteers and psychiatric patients.

Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway

AbstractLarge granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-β transcripts than purified normal CD8+ T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

The hemostatic profile of recombinant activated factor VII. Can low concentrations stop bleeding in off-label indications?

BackgroundHigh concentrations of recombinant activated factor VII (rFVIIa) can stop bleeding in hemophilic patients. However the rFVIIa dose needed for stopping haemhorrage in off-label indications is unknown. Since thrombin is the main hemostatic agent, this study investigated the effect of rFVIIa and tissue factor (TF) on thrombin generation (TG) in vitro.MethodsLag time (LT), time to peak (TTP), peak TG (PTG), and area under the curve after 35 min (AUCo-35 min) with the calibrated automated thrombography was used to evaluate TG. TG was assayed in platelet-rich plasma (PRP) samples from 29 healthy volunteers under basal conditions and after platelet stimulation with 5.0 μg/ml, 2.6 μg/ml, 0.5 μg/ml, 0.25 μg/ml, and 0.125 μg/ml rFVIIa alone and in normal platelet-poor plasma (PPP) samples from 22 healthy volunteers, rFVIIa in combination with various concentrations of TF (5.0, 2.5, 1.25 and 0.5 pM).ResultsIn PRP activated by rFVIIa, there was a statistically significant increase in TG compared to basal values. A significant TF dose-dependent shortening of LT and increased PTG and AUCo→35 min were obtained in PPP. The addition of rFVIIa increased the effect of TF in shorting the LT and increasing the AUCo→35 min with no effect on PTG but were independent of rFVIIa concentration.ConclusionLow concentrations of rFVIIa were sufficient to form enough thrombin in normal PRP or in PPP when combined with TF, and suggest low concentrations for normalizing hemostasis in off-label indications.

A multicolor flow cytometric assay for measurement of platelet-derived microparticles

Introduction Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for “bedside” analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements. Materials and Methods PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments. Results Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100–700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were < 10%, and the variation between two cytometers in two different laboratories was < 5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls ( p < 0.001). Conclusions The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples.

Blood Clotting by Low-Temperature Air Plasma

In this paper, the feasibility and effectiveness of using an air-plasma torch to clot blood are studied. The emission spectroscopy and temperature measurement of the torch show that the torch produces abundant atomic oxygen in the plasma effluent. Anticoagulated whole-blood samples (10 mulscr ) were tested for two cases: 1) sample exposed to the plasma effluent and 2) sample exposed to a heated airflow; in both cases, the increase of the sample temperature was controlled to be about the same. No indication of blood coagulation was observed in the second case; on the other hand, in the first case when the blood sample was exposed directly to the plasma effluent, a shell was formed on the surface of the blood sample. The experimental results demonstrated that this plasma torch could clot a blood sample via a nonthermal mechanism. The dependence of the degree of blood clotting on the atomic-oxygen flux was demonstrated by varying the exposure distance. Experimental results also showed that blood coagulation was seen in the treated platelet-rich plasma sample but not in the treated platelet-poor plasma sample. Moreover, plasma treatment with multiple short exposures was shown as more effective in clotting blood than that of applying a continuous exposure with the same total exposure time.

Changes of Platelet and Endothelial Microparticles in Patients with Venous Thromboembolism

Background: Microparticles (MP) are small membrane vesicles released from a variety of cells including platelet, leukocyte, endothelial cell which mediate intravascular coagulation. In this study, the plasma levels of total microparticle (TMP), platelet microparticle (PMP) and endothelial microparticle (EMP) in patients with venous thromboembolism (VTE) were investigated to test their clinical utilities.Methods: Subjects were divided to 3 groups consisted of 119 patients with VTE, 72 cancer without VTE and 93 healthy controls. MP were assayed by flow-cytometry using non-washed, frozen platelet-poor plasma samples. TMP, PMP and EMP were identified using antibodies against Annexin V, CD41a, and CD144 respectively.Results: The mean levels of plasma TMP, PMP, but not EMP in VTE without caner group (n=77) were significantly increased compared with those in healthy controls (n=93); 1972.9/μl vs 917.0/μl, p=0.001; 1496.6/μl, 586.5/μl, p=0.001; 108.0/μl vs 97.8/μl, p=0.321). In the VTE without cancer group, the mean levels of plasma TMP, PMP, but not EMP in newly diagnosed VTE group (n=59) were significantly increased compared with previously diagnosed VTE group (n=18); 2242.5/μl vs 1089.1/μl, p=0.008; 1728.6/μl vs 735.9/μl, p=0.005; 101.8/μl vs 84.6/μl, p=0.885). Mean peak thrombin generations in VTE samples (n=9) were increased in platelet poor plasma (385.2 nM vs 163.1 nM, p=0.002) but not in microparticle free plasma (MFP) (86.2 nM vs 22.6 nM, p=NS) as compared with those of controls (n=10) suggesting the thombin generation is originated from MP of plasma. But, the mean levels of plasma TMP, PMP, but not EMP in VTE with caner group (n=42) were not increased compared with those in cancer controls without VTE (n=72); 2158.1/μl vs 1644.2/μl, p=0.094; 1637.2/μl vs 1246.8/μl, p=0.168; 86.0/μl vs86.0/μl, p=0.996). The plasma levels of B-TG were well correlated with those of PMP. The levels of plasma TMP, PMP, and EMP did not show any correlation with the level of D-dimer.Conclusion: The plasma levels of TMP and PMP are significantly increased in VTE patients and the levels are normalized after anticoagulation, suggesting MP may be used as surrogate markers for monitoring of VTE.

Use of the Thrombin Generation Test to Evaluate Response to Treatment With Recombinant Activated Factor VII

F l i ( ecombinant activated factor VII (rFVIIa; NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) represents an effecive treatment for hemophilia patients with inhibitors. Howver, there is no consensus on the optimal dosing regimen. sing the recommended dose of 90 g/kg, hemostatic effiacy is obtained in 85% of cases. The efficacy and safety of a ingle high dose of rFVIIa (270 g/kg) also have been demnstrated.1–3 As the final product generated by rFVIIa is hrombin, the thrombin generation test (TGT) could theoretcally be used to monitor individual responses to rFVIIa by ach hemophilia patient with an inhibitor. In this study, we assessed the role of TGT in monitoring he response to rFVIIa in three patients with severe hemohilia A with an inhibitor. Thrombin generation (TG) curves ere obtained in platelet-poor plasma (PPP) and platelet-rich lasma (PRP) samples spiked in vitro with rFVIIa at doses of 5, 90, 120, 160, 200, 240, and 270 g/kg. The ex vivo TG apacity was measured before and 15 minutes, 1 hour, and 2 ours after the infusion of a therapeutic dose of rFVIIa. The in vitro results showed that the addition of rFVIIa ose-dependently increased TG capacity. The comparison of n vitro and ex vivo results suggested that in vitro TG spiking xperiments could be helpful in predicting the individual inimal effective dose of rFVIIa. The ex vivo results showed n important increase in TG capacity following an injection f rFVIIa with a variable rate of correction in the three paients studied. These data are in agreement with clinical nowledge showing an individual response to rFVIIa. In adition, our results clearly demonstrate that the monitoring of FVIIa by TGT using PPP underestimated the hemostatic ef-

Evaluation of the REG1 Anticoagulant System Using Thrombelastography and Thrombin Generation Assay.

Background: The REG1 system (Regado Biosciences, Inc. Durham, NC) is a novel anticoagulant system which comprises RB006 (drug) and RB007 (antidote). The aptamer, RB006 selectively binds to FIXa and blocks factor Xa generation. RB007 is a complementary ligand that selectively binds to RB006, and reverses its anticoagulant effect (1). Phase 1 testing demonstrated a clear pharmacodynamic dose response to RB006 in plasma coagulation assays (1), but further work is needed to understand the pharmacodynamic response to the REG1 system in whole blood assays. Therefore, we evaluated the effects of RB006 and RB007 alone and in combination using activated partial thromboplastin time (APTT), viscoelastic (TEG®, Haemoscope, IL) and thrombin generation assay (Thrombinoscope™, Synapse BV).Methods: After IRB approval, blood samples were collected from 4 consented healthy volunteers into 3.2% citrate tubes. For APTT and TEG testing blood was placed in thirteen 2 ml Eppendorf tubes. Tube one had 20 μl of saline added and served as a control. The remaining 12 tubes were divided into 3 groups. Group1: RB006 (final concentrations 3, 6, 12, 18 and 24 μg/ml); Group 2: RB007 (final concentrations 6, 12, 24, and 48 μg/ml) and Group 3: combination of both agents at a weight:weight ratio 2 to 1 for RB007:RB006 (final concentrations as above). All testing was performed within 3-hour of blood draw. APTT was done using Hemochron Jr® (ITC, NJ) instrument in recalcified whole blood. TEG was peformed in recalcified whole blood, 360 μl activated with 2 nM thrombin. For Thrombinoscope, whole blood was centrifuged at 2000 x g for 15 min to obtain platelet poor plasma (PPP). PPP samples were prepared to contain the same concentrations of RB006, RB007, and combination of both agents as described in whole blood samples. Thrombin generation analyses were performed using microplate format using diluted Actin (Dade Behring, Marburg, Germany) as a trigger (2).Results: RB006 dose dependently increased APTT (Table 1). Increasing concentrations of RB006 progressively prolonged onset, and decreased the rate of thrombus formation on TEG (Figure1A). On Thrombinoscope, RB006 dose dependently delayed lag time and decreased peak thrombin generation (Table 1, Figure1B). All the parameters of aPTT, TEG and thrombin generation returned back to control values when combination of aptamer-anti-aptamer was tested. RB007 alone had no effects on any of the tests performed.Conclusion: Along with conventional point of care APTT testing TEG and Thrombinoscope methodologies can be very useful in monitoring the anticoagulation effects of aptamer, RB006, and its reversal with anti-aptamer, RB007.Effects of Aptamer, RB006, on aPTT and Thrombinoscope lag time/peak thrombinRB006μg/mlAPTT secLag time minPeak thrombin nM0 (control)51.3 ± 1.010.8 ± 0.3244 ± 16.2395.3 ± 3.014.2 ± 0.9173 ± 22.96152 ± 10.018.8 ± 0.5145 ± 23.012183 ± 4.324.1 ± 1.1105 ± 11.018238 ± 15.326.3 ± 2.392.3 ± 7.324257 ± 11.331.3 ± 3.888.2 ± 8.3Data shown as mean ± SD [Display omitted]

Patients with multiple myeloma treated with thalidomide: evaluation of clinical parameters, cytokines, angiogenic markers, mast cells and marrow CD57+ cytotoxic T cells as predictors of outcome

In vitro studies suggest that thalidomide has an immunoregulatory role and alters the marrow microenvironment. We assessed laboratory and clinical parameters in patients with myeloma treated with thalidomide as potential prognostic markers and looked for changes with therapy. Seventy-five patients with relapsed/refractory myeloma received thalidomide in a phase II trial. Serial samples of platelet-poor plasma and bone marrow were tested for angiogenic cytokines including vascular endothelial growth factor (VEGF), marrow microvessel-density (MVD), mast cells and CD57+ cell expression. The effects of these parameters on response rate (RR), progression-free survival (PFS) and overall survival (OS) were analyzed. Elevated baseline VEGF predicted for a superior RR (p=0.018) and PFS. Elevated CD57+ cells also predicted superior PFS (p=0.012). MVD did not predict for RR, PFS or OS, but MVD and VEGF fell significantly in responders. Multivariate analysis identified that inferior OS was associated with age >65 years (p=0.017), raised lactate dehydrogenase (p=0.001), raised hepatocyte growth factor levels (p=0.012) and low pre-treatment CD57+ cells (p<0.001). Our findings support the suggestion that thalidomide has anti-angiogenic and immunomodulatory effects in myeloma. The preferred method for assessing angiogenesis is plasma VEGF levels and the assessment of CD57+ cells for patients with myeloma receiving novel immunomodulatory drugs should be further investigated.

Effects of Multimerin 1 on Platelet and Plasma FV as Determined by Calibrated Automated Thrombography.

Multimerin 1 (MMRN1) is a protein that is not found in plasma and that is complexed with factor V (FV) in platelet α-granules. Following thrombin activation, platelet FV and MMRN1 are released, and FV is converted to Va, promoting its dissociation from MMRN1. Recent studies identifying functional overlap between FV/Va binding sites for phospholipids and MMRN1, and unanswered questions about the functional consequences of MMRN1 on platelet FV function, led us to compare how MMRN1 modulates platelet and plasma FV procoagulant functions using calibrated automated thrombograms (CAT). The effects of platelet and exogenous MMRN1 on FV activity were evaluated using samples of platelet poor human plasma or platelet extracts resuspended in FV deficient plasma to equivalent final FV antigen concentrations. Reactions were triggered with a commercial reagent containing saturating concentrations of tissue factor and phospholipids (60:20:20 phosphatidylcholine: phosphatidylserine: phosphatidylethanolamine). These assays were also used to compare the functional properties of FV in MMRN1-immunodepleted and sham-depleted fractions of a soluble platelet protein extract (prepared with calpain inhibitors). The effects of exogenous MMRN1 on platelet-dependent thrombin generation were also evaluated using normal platelets resuspended in FV deficient plasma. In contrast to plasma, platelet fractions (with an equivalent FV antigen content) consistently showed lower peak and total endogenous thrombin potential. Addition of 17 fold molar excess of exogenous MMRN1 (which represents the physiologic ratio of MMRN1 to FV in platelets) to plasma, or to MMRN1 depleted platelet protein extracts, induced similar reductions in thrombin generation, manifested by increases in lag times (p<0.05), and reductions in peak (p<0.05), and total thrombin generation (p<0.05). Higher concentrations of exogenous MMRN1 caused further attenuation. Analogous, dose-dependent, inhibitory effects of MMRN1 were observed on thrombin generation by normal platelets in FV deficient plasma. Further investigations of the thrombin generating potential of platelet FV were done using sham and MMRN1-depleted platelet extracts. While exogenous MMRN1 inhibited platelet FV dependent thrombin generation, sham-depleted platelet FV (containing MMRN1) consistently showed higher thrombin generation than the same quantity of FV in MMRN1-depleted platelet extracts, characterized by shorter lag times (p<0.05), higher peak thrombin generation (p<0.05), and increased endogenous thrombin potential (p<0.05). Although the sham-depleted fraction had a similar reduced mobility to the MMRN1 depleted platelet FV, it contained very large forms of platelet FV, linked to MMRN1 via the human FV B-domain cysteine. These data indicate that the pool of platelet FV associated with MMRN1 in vivo has procoagulant function and that MMRN1 has inhibitory effects on thrombin generation. The observed differences in the procoagulant properties of platelet and plasma FV in CAT assays likely result from a partial inhibition of platelet FV by MMRN1 that may be counterbalanced by partial platelet FV activation. The paradoxical effects of MMRN1 depletion on the procoagulant function of platelet FV suggests MMRN1 may have other influences that help preserve platelet FV function in vitro, and possibly also in vivo. The MMRN1 liberated by activated platelets and endothelium in vivo could have local modulating effects on thrombin generation.

Influences of Blood Sample Processing on Low–Molecular-Weight Proteome Identified by Surface-Enhanced Laser Desorption/Ionization Mass Spectrometry

Profiling approaches in proteomics, such as surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, are used in disease marker discovery. The aim of this study was to investigate the potential influence of selected preanalytical factors on the results obtained. Plasma samples anticoagulated with EDTA, citrate, or heparin, and serum samples from healthy volunteers were profiled by SELDI on CM10, immobilized metal affinity capture (IMAC) array with copper, and H50 chip surfaces. Using linear mixed-effects models, we examined the influence of elapsed time between venipuncture and sample separation (immediate to 24 h) and the type of serum tube used (Greiner Vacuette activator, gel serum separator, or plain tubes). We analyzed purified platelets, as well as platelet-poor and platelet-rich plasma samples treated with calcium and/or thrombin to determine the platelet contribution, directly or via the clotting process, to the profiles generated. We then used cluster analysis to identify samples with similar peak profiles. Different plasma types and sera could be distinguished on the basis of cluster analyses of their spectral profiles. Elapsed time between venipuncture and separation of plasma and serum from blood samples altered the profiles obtained, particularly for serum samples and particularly on IMAC chips. The type of serum collection tube also affected the profiles because of differences in clotting time. In vitro manipulation of platelets revealed that specific peaks in IMAC profiles of serum appeared to be derived directly from platelets. Several other peaks, including some of those exhibiting time-dependent changes, arose during the clotting process. Preanalytical variables, such as sample handling, can markedly influence results.

The Effects of Platelet Count on Clot Retraction and Tissue Plasminogen Activator-Induced Fibrinolysis on Thrombelastography

Clot retraction and fibrinolysis may present as a decrease in amplitude on thrombelastography (TEG). The former represents normal or hyperactive platelet function, and the latter represents a fibrinolytic state. It is important to distinguish clot retraction from fibrinolysis because the treatment of each condition is different. To distinguish between these phenomena, we performed TEG with platelet-poor plasma (PPP) and platelet-rich plasma (PRP) with an increasing platelet count (range, 50-1200 x 10(9)/L) with or without abciximab. Maximum amplitude (MA) and the percentage decrease of amplitude at 30 and 60 min after MA were examined for each sample. Blood samples to which tissue plasminogen activator (tPA) was added served as positive controls for fibrinolysis. Morphological changes of clots and D-dimer levels were also examined. With higher platelet counts, the percentage decrease of amplitude after MA increased significantly at 30 and 60 min, but not in the abciximab samples. Morphological changes of clots have shown clot retraction in PRP, but not in PPP or PRP pretreated with abciximab. D-dimer levels increased only in samples to which tPA was added, but not in native PPP or PRP samples. In conclusion, we have shown that the decrease in amplitude at 30 and 60 min can be due to platelet-mediated clot retraction and can be attenuated by sample pretreatment with abciximab, which interrupts platelet-fibrin(ogen) binding.

Effect of FV-Leiden Mutation and Antiphospholipid Syndrome on Thethrombin GenerationTest.

Introduction: The incidence of Factor V Leiden mutation (Arg 506 Gln) is significantly high in patients with venous and arterial thrombosis. In patients with antiphospholipid syndrome (APS), the FVL mutation may play a major role in the occurrence of thrombosis. The aim of this study was to demonstrate that an increased Endogenous Thrombin Potential (ETP) may be an important mechanism for the thrombotic risk associated with FVL and / or APS and to know if the FVL mutation would increase the risk of thrombosis in patients with APS. Additionally, we tested if fluorometric determination of ETP( area under the curve), thrombin generation velocity (PEAK) and TIME TO PEAK (time from starting to reaching the peak), is suitable to determine changes in haemostatic parameters.Methods and patients: We measured the thrombin generation in 120 patients (67F, 53M) with a median age of 40 year categorized in 3 groups of 40 patients each:(1) either heterozygous or homozygous FVL mutation (14F, 26M of whom 29 were heterozygous and 11 were homozygous) (2) presence of antiphospholipid antibodies (aPL) (20F, 20M) and (3) a combination of both (33F,7M). Three characterizing thrombin generation parameters were measured: ETP, PEAK and TIME TO PEAK. Platelet poor plasma samples (PPP) were derived from citrated blood. In a microtiter plate, we used different concentrations of TF with phospholipids after adding PRP(platelet poor plasma) and buffer. The reaction was started after adding substrate solution. Fluorometer was the Fluoroskan Ascent Type 374.Results: Using four concentrations (Innovin dilutions 1:600, 1:6.000, 1:50.000 and 1:500.000) of TF, the following results (ETP units = relative fluorescence units or RFU; PEAK units: RFU/min; TIME TO PEAK units = min) were obtained for the 3 groups:•For all patients TF 1:600 median ETP, PEAK and TIME TO PEAK were taken as 100 % because less dilution gives no further increase resp. decrease in these parameters•Patients with aPL: median ETP decreased from 100 % to 36,6 % (lowest TF concentration), PEAK to 34,4 % and TIME TO PEAK increased to 218 %.•Patients with FVL median ETP decreased from 100 % to 2,64 % (lowest TF concentration), PEAK to 9 % and TIME TO PEAK increased to 282 %.•Patients with aPl and FVL => median ETP decreased from 100 % to 33 % (lowest TF concentration), PEAK to 30,8 % and TIME TO PEAK increased to 143 %.While there was no detectable thrombin generation in aPL patients in low concentration of TF, at the two highest concentrations the thrombin generation was comparable to same concentrations in the haemostatically normal control samples(data not shown). For those affected by FVL and aPL, the threshold of detectable thrombin generation was even shifted to lower concentrations of TF, regarding a “residual activity” of about 33 % for ETP and PEAK, as for patients with FVL. In patients with APS, TF 1:50.000 caused a thrombin generation comparable to TF 1:6.000 in haemostatically normal controls, showing a shift in coagulation factor reactability. In a physiological environment this could indicate a stronger haemostatic reaction to minor vascular lesions in patients affected by factor V Leiden mutation than in those without it.Conclusion: A thrombin generation assay utilizing lower concentrations of TF as coagulation initiating agent could be usefully performed to assess thrombophilic states due to coagulation factor mutations and /or presence of antiphospholipid antibody.

Prospective study of circulating angiogenic markers in prostate-specific antigen (PSA)-stable and PSA-progressive hormone-sensitive advanced prostate cancer

Objectives To prospectively describe and compare circulating vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in two groups of advanced prostate cancer patients undergoing androgen deprivation. The first patient group (n = 21) consisted of patients with stable serum prostate-specific antigen (PSA) and the second group (n = 20) consisted of patients with a rising serum PSA during androgen deprivation. Methods Patients with diabetes or active heart disease or those receiving anticoagulants were excluded. Circulating VEGF and bFGF were measured in platelet-poor plasma. bFGF was also measured in urine. Platelet factor 4 protein (PF4) assays were performed to evaluate platelet activity in platelet-poor plasma samples. Commercially available enzyme-linked immunosorbent assay kits were used for all assays, and all tests were performed in duplicate. Results The median age of this study population was 75 years (range 58 to 85). Median plasma VEGF measured in the PSA-stable group was 801.5 pg/mL and in the PSA-rising group was 655.5 pg/mL ( P = 0.464). Circulating bFGF was undetectable in plasma, but 4 patients in the PSA-stable group had measurable urine levels. Platelet-poor plasma PF4 assays in all patients were less than 3 IU/mL (normal range 0 to 10). Conclusions Our pilot study suggests elevated plasma VEGF levels in advanced prostate cancer do not increase during failure of androgen deprivation therapy. Most of the advanced cancer patients in this study expressed plasma VEGF. This suggests its potential role as a surrogate marker for response assessment during antiangiogenic therapy in this stage.

Coagulative System Activation and Fibrinolytic System Inhibition Activities Arise From Tumoral Draining Vein in Colon Carcinoma

Hypercoagulability and activation/inhibition of the fibrinolytic system have been observed in abdominal cancer surgery. Because surgery itself and also neoplastic diseases are associated with these situations, a method for separating the origin of these two processes was designed. Eighteen patients with colon cancer who underwent a surgical procedure were studied: Immediately before surgery blood was taken from a peripheral vein. During the surgical procedure, before the exclusion of tumoral tissue from general circulation and at the same time of a second peripheral vein blood sample, a blood sample was taken from the main tumoral draining vein. Platelet-poor plasma samples were aliquoted and stored at −72 °C, ready for analytical procedures. Results: A moderate activation of the fibrinolytic system during surgery was observed, expressed by elevation of tissue plasminogen activator (t-PA) ( P<.05) and D-dimer (DD) ( P<.05) levels, without changes in fibrinogen (FG), plasminogen (PG) or antiplasmin (AP) levels. There were no modifications in antithrombin III (AT-III) and protein C (PC) levels. In the tumoral draining vein samples, there was a high elevation of levels of thrombin–antithrombin III complexes (TAT) ( P<.001) and PAI-1 ( P<.01), compared with the second sample peripheral vein. There was no difference between peripheral and tumoral vein sample levels of AT-III, PC, FG, DD, PG and AP. Conclusions: The tumour itself is the origin of hypercoagulability (TAT) and fibrinolytic system inhibition (PAI-1); the surgical procedure elicits an evident though moderate activation of the fibrinolytic system (t-PA and DD elevation).

Near-site monitoring of the antiplatelet drug abciximab using the hemodyne analyzer and modified thrombelastograph

Objective: This investigation examines the hypothesis that the antiplatelet effect of abciximab and its reversal can be monitored using the Hemodyne (Hemodyne, Inc, Midlothian, VA) analyzer and modified Thrombelastograph (Haemoscope, Skokie, IL). Design: In vitro dose-response and reversal study. Setting: Anesthesia Research (Dallas, TX) and Special Studies Coagulation Laboratories (Washington, DC). Participants: Nine healthy volunteers. Interventions: The addition of increasing concentrations of abciximab, 0 to 10 μg/mL, and purified fibrinogen, 50 to 400 mg/dL. The reversal of abciximab, 4 μg/mL, with the addition of fresh platelet-rich plasma (PRP) sufficient to increase the platelet concentration by approximately 10%. Measurements and Main Results: Platelet aggregation and platelet contractile force using the Hemodyne analyzer were used as platelet-specific measurements. The Thrombelastograph maximum amplitude (MA) for platelets (MA PLT) was calculated by subtracting the MA from a platelet-poor plasma (PPP) sample (MA ppp) determined in one thromboelastography well from that of whole-blood MA (MA WB) run simultaneously in the second thromboelastography well. The addition of abciximab, 0 to 10 μg/mL, resulted in significant concentration-dependent reductions in platelet aggregation ( p < 0.001), platelet contractile force ( p < 0.001), and MA PLT ( p < 0.001). Platelet contractile force ( p < 0.03) and MA PLT ( p < 0.05) were significantly more responsive than MA WB to the effect of abciximab, 4 μg/mL, and its reversal with the addition of fresh PRP. Purified fibrinogen concentration directly correlated with thromboelastography MA ( r s = 0.97; p < 0.001), yet had no effect on platelet contractile force. The addition of abciximab had no measurable influence on the MA PPP. Conclusion: This in vitro study suggests that the Hemodyne analyzer and modified Thrombelastograph might be clinically useful methods to monitor the platelet inhibitory effects of agents such as abciximab.

DOSE DEPENDENT EFFECTS OF c7E3 Fab ON MODIFIED THROMBOELASTOGRAPHY IN HEALTHY VOLUNTEERS

S64 Introduction: A "point of care" platelet function monitor has yet to be established. Although thromboelastography (TEG) has been used clinically, [1] it lacks platelet specificity and requires 30-45 minutes to complete. Modified TEG has been shown to increase the responsiveness of this assay to platelet receptor antagonism and requires only 15 minutes to complete. [2,3] We hypothesized that: 1) modified TEG maximum amplitude (mTEG:MA) is decreased in a dose-dependent manner when exposed to the platelet glycoprotein receptor antagonist c7E3 Fab (REOPRO[trade mark sign]); 2) alterations in fibrinogen concentration significantly influences the TEG:MA. Methods: Following IRB approval and informed consent, whole blood (WB), platelet-rich and platelet poor plasma (PPP) samples from 5 healthy volunteers were used to measure the effects of c7E3 Fab (0-10 [micro sign]g/mL) on the TEG (Haemoscope Corp., Skokie, IL), platelet contractile force (PCF)(Hemodyne Corp., Richmond, VA) and aggregometry (ADP). mTEG:MA was calculated by subtracting MA from PPP from the WB MA. The TEG:MA of purified fibrinogen (50-400mg/dL; Sigma Chem.) was also performed in duplicate. ANOVA and linear regression was used to analyze the effects of c7E3 Fab and fibrinogen on the TEG:MA. P-values <or=to 0.01 were significant. Results: Figure 1 demonstrates the dose-dependent effects of c7E3 Fab. Figure 2 illustrates the influence of fibrinogen on the MAppp. Linear regression analysis of purified fibrinogen is inset in Figure 2. Figure 3 lists the remaining coagulation data for the volunteers.Figure 1Figure 2Figure 3Discussion: This is the first reported use of modified TEG demonstrating the dose-dependent effects of c7E3 Fab on platelet function in healthy volunteers. The influence of fibrinogen on the TEG:MA can be substantial. This is illustrated by the generation of a "normal" TEG:MA using purified fibrinogen (Figure 2). Although modified TEG was not as responsive to clinical doses of c7E3 Fab (4[micro sign]/mL) as platelet contractile force or aggregometry (Figure 1), it is the only commercially available technology at this time.

High-performance liquid chromatographic determination of taurine in human plasma using pre-column extraction and derivatization

Plasma samples (100 μl) were treated with 150 μl of acetonitrile and centrifuged at 5800g for 10 min and 50 μl of 10 mM borate buffer (pH 9.2) were added to the supernatant solution. This was followed by the addition of a 50 μl aliquot of 5 mM fluorescamine in acetonitrile and immediate vortex mixing. A 20 μl sample was injected on to a reversed-phase HPLC system using a Bondclone C-18 10 μm analytical column (300 mm × 3.9 mm). The mobile phase was tetrahydrofuran—acetonitrile—phosphate buffer (15 mM, pH 3.5) (4:24:72, v/v/v). The taurine derivative was detected by measuring the UV absorbance of 385 nm. Platelet-poor plasma samples were spiked with known amounts of taurine and inter- and intra-assay calibration curves were obtained. The method was applied to the determination of taurine in platelet-rich plasma.

Euglobulin lysis time in fresh and stored samples.

Euglobulin lysis time is a global test for the study of fibrinolysis. The aim of this study was to evaluate the influence of storage of plasma and euglobulin precipitates on euglobulin lysis time, by testing samples stored in different conditions. In 20 healthy subjects, euglobulin lysis time was measured by (1) euglobulin precipitates prepared within 90 minutes from blood withdrawal (reference euglobulin lysis time); (2) euglobulin precipitates obtained from platelet-poor plasma stored for 24 hours at either -80 degrees C or at -20 degrees C; (3) euglobulin precipitates frozen for 24 hours at either -80 degrees C or at -20 degrees C; (4) euglobulin precipitates dissolved in Owren's buffer and frozen for 24 hours at either -80 degrees C or at -20 degrees C. Euglobulin lysis time measured on euglobulin precipitates dissolved in Owren's buffer and stored at -20 degrees C and at -80 degrees C, and euglobulin lysis time measured on platelet-poor plasma stored at -20 degrees C were significantly longer than the reference euglobulin lysis time (at least P < .05). On the contrary, no changes were observed in euglobulin lysis time measured on platelet-poor plasma stored at -80 degrees C, and on euglobulin precipitates undissolved and stored at -20 degrees C and at -80 degrees C versus reference euglobulin lysis time. The pattern was similar in samples obtained both before and after venous occlusion. These data indicate that the freezing of samples of platelet-poor plasma or euglobulin precipitates at -80 degrees C and of euglobulin precipitates at -20 degrees C makes the simultaneous determination of a large number of samples collected at different times the previous day possible.