Inverse relationship between the severity of gingivitis and platelet reactivity in stable angina pectoris.
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Aged↗
Biomedical subjects
T L Lindahl
Publications and source records attributed to T L Lindahl.
Evaluation of platelet function, a method comparison.
Platelet function can be studied using many different methods why it is of interest to understand how data from different assays relate to each other. In the present study we compare two methods suitable for screening purposes with two established although laborious methods, impedance aggregometry and platelet-rich plasma (PRP) aggregation. The alternative assays tested were: (i) exposure of active alphaIIbbeta3, in diluted whole blood and (ii) whole blood aggregation assessed by residual platelet counting. The fibrinogen receptor activation assay was found to have the lower variability, higher sensitivity to ADP, and higher signal to noise ratio compared with residual platelet counting. The sensitivity and response profile of the fibrinogen receptor activation assay and residual platelet counting were more similar to PRP aggregation than to impedance aggregometry, whereas impedance aggregometry displayed lower sensitivity to ADP. The two alternative assays correlated well with PRP aggregation as well as with each other. The fibrinogen receptor activation assay displayed the highest potency for AR-C69931MX, possibly due to a lower protein content compared with residual platelet counting. The two studied assays compare well with the more established assays, and are thus both good alternatives for platelet function testing and evaluation of new potential platelet antagonists.
Adenosine Diphosphate↗
Main haplotypes and mutational analysis of vitamin K epoxide reductase (VKORC1) in a Swedish population: a retrospective analysis of case records.
BACKGROUND: Vitamin K epoxide reductase (VKORC1) is the site of inhibition by coumarins. Several reports have shown that mutations in the gene encoding VKORC1 affect the sensitivity of the enzyme for warfarin. Recently, three main haplotypes of VKORC1; *2, *3 and *4 have been observed, that explain most of the genetic variability in warfarin dose among Caucasians. OBJECTIVES: We have investigated the main haplotypes of the VKORC1 gene in a Swedish population. Additional objective was to screen the studied population for mutations in the coding region of VKORC1 gene. PATIENTS/METHODS: Warfarin doses and plasma S- and R-warfarin of 98 patients [with a target International Normalized Ratio (INR) of 2.0-3.0] have been correlated to VKORC1 haplotypes. Controls of 180 healthy individuals have also been haplotyped. Furthermore, a retrospective analysis of case records was performed to find any evidence indicating influence of VKORC1 haplotypes on warfarin response in the first 4 weeks (initiation phase) and the latest 12 months of warfarin treatment. RESULTS AND CONCLUSIONS: Our result shows that VKORC1*2 is the most important haplotype for warfarin dosage. Patients with VKORC1*2 haplotype had more frequent visits than patients with VKORC1*3 or *4 haplotypes, higher coefficient of variation (CV) of prothrombin time-INR and higher percentage of INR values outside the therapeutic interval (i.e. 2.0-3.0) than patients with VKORC1*3 or *4 haplotypes. Also, there was a statistically significant difference in warfarin dose (P < 0.001) and R-warfarin plasma levels (P < 0.01) between VKORC1*2 and VKORC1*3 or 4 haplotypes. Patients with VKORC1*2 haplotype seem to require much lower warfarin doses than other patients.
Adult↗
Guidelines on preparation, certification, and use of certified plasmas for ISI calibration and INR determination--a rebuttal.
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Calibration↗
Identification of low-density platelet populations with increased reactivity and elevated alpha-granule content.
The present study examines biochemical and functional characteristics of platelet density subpopulations together with their ability to mobilise intracellular fibrinogen when activated. Platelets from three healthy volunteers were investigated. The total platelet population was separated according to density in a linear Percoll gradient in a plasma-free milieu containing EDTA that binds soluble Ca(2+). Subsequently, platelets from each individual were divided according to density into 11 or 12 aliquots. In all fractions, we determined platelet count, intracellular P-selectin and the ADP-evoked platelet fibrinogen binding as a measure of platelet reactivity together with the platelet dense body content. The work demonstrates that platelets use stored intracellular fibrinogen when activated. It also shows that the platelet-fibrinogen binding can be initiated in a surrounding depleted of Ca(2+) and fibrinogen. Moreover, the study demonstrates subpopulations of light platelets having increased reactivity and more alpha-granules but less dense bodies. The biological significance of the findings needs to be elucidated.
Adenosine Diphosphate↗
A new flow cytometric method for measurement of von Willebrand factor activity.
BACKGROUND: Patients with von Willebrand's disease may have normal levels of von Willebrand factor (VWF) antigen. It is therefore important to measure not only the antigen concentration but also the VWF activity. The most widely used method for measurement of VWF activity is the ristocetin cofactor assay (VWF:RCo), which is still crucial for the laboratory diagnosis of von Willebrand's disease (VWD). However, VWF:RCo has low precision, poor inter-laboratory reproducibility and requires an aggregometer. Many routine laboratories are not equipped with aggregometers but have flow cytometers instead. METHODS: In this study a simple, precise and rapid flow cytometric assay was developed for the determination of von Willebrand factor activity, utilizing formalin-fixed platelets, fluorescein isothiocyanate-conjugated chicken anti-VWF antibodies (Fab-fragments) and phycoerythrine-conjugated anti-GPIIb/IIIa antibodies. RESULTS: In samples from healthy controls and from patients with von Willebrand disease type 1, the flow cytometry assay showed good correlation with the VWF:RCo assay (r2 = 0.69) and the VWF antigen assays (r2 = 0.83), which was better than the correlation between the VWF:RCo assay and VWF antigen assays (r2 = 0.72). The flow cytometry method had good within-assay and total precision, C.V. 4.2%, and C.V. 7.5%, at a mean concentration of 0.40 IU/mL, respectively. Results obtained with the flow cytometric method on samples from two patients with von Willebrand disease 2B were lower than those obtained with the antigen method in accordance with the diagnosis. CONCLUSION: The accuracy and precision of the von Willebrand activity assay may be improved if a flow cytometer is utilized for measurement of the impact of ristocetin on binding of VWF to formalin-fixed platelets instead of measuring agglutination utilizing an aggregometer. In addition, our flow cytometric method assay enables measurement of von Willebrand factor activity at many more hospitals than was previously possible with the traditional ristocetin cofactor platelet aggregometry assay, and this trend is likely to increase in the future when routine hematological instruments are equipped with built-in flow cytometers.
Antibodies↗
Clotting time by free oscillation rheometry and visual inspection and a viscoelastic description of the clotting phenomenon.
An automated procedure for determination of clotting time in whole blood was validated by direct comparison with the reference method, visual clotting time determination. The procedure was based on a 10 Hz free oscillation rheometer (FOR) of our design, the ReoRox 4. Recalcified citrated blood samples (n = 30), clotting in the range 4 to 20 min, were used in the validation. Every 30 s of the analysis, as the change in stiffness (deltaG*) of the sample was monitored by FOR, the sample cup was shortly removed from the FOR and its contents inspected for first signs of clotting, i.e. visual clotting time determination. Various FOR clotting criteria were attempted. Best correlation to visual clotting time was found when deltaG* reached 0.01 Pa, which yielded linear regression slope, intercept and r2 of 0.98, 0.09 min and 0.98, respectively. For comparison, six plasma samples were analyzed in the same way and gave almost the same results. The accuracy of the FOR determinations was checked by also analyzing, in parallel, portions of the sample with a conventional oscillation rheometer, a Bohlin VOR. The rationale is given for preferring deltaG* over G* as a FOR monitoring function in coagulation tests and for including median filtration of the primary FOR data. An extension of the FOR theory to include deltaG* and evidence in support of inhomogeneous blood clotting are also given.
Blood Coagulation↗
Individual variations of platelet inhibition after loading doses of clopidogrel.
OBJECTIVE: To investigate individual variations of platelet inhibition after clopidogrel-loading doses. SETTING: Department of Cardiology, Linköping University Hospital, Linköping, Sweden. SUBJECTS: Individuals with stable angina pectoris (n = 18) subject to percutaneous coronary interventions (PCI) and subsequent stenting were investigated. METHODS AND EXPERIMENTAL PROTOCOL: A 300-mg clopidogrel loading dose was administrated immediately after stenting (day 1) followed by an additional 75 mg clopidogrel after 24 h (day 2). The ADP-evoked platelet fibrinogen binding was analysed to estimate platelet reactivity immediately before angiography and on day 2. A flow cytometry technique was used with two ADP solutions (final concentrations 0.6 and 1.7 micromol L-1) employed as platelet activating agents. Soluble P-selectin was used as a marker of platelet activity. RESULTS: When using 1.7 micromol L-1 ADP to activate platelets four individuals had a strong inhibition (i.e. platelet reactivity <10% of the day 1-value day 2). In contrast, five patients demonstrated a weak inhibition (i.e. platelet reactivity >60% of the day 1-value day 2). Similar results were obtained when using 0.6 micromol L-1 ADP as a platelet-activating agent. Clopidogrel, however, fails to suppress platelet activity as estimated from soluble P-selectin. CONCLUSIONS: Clopidogrel evoked platelet inhibition exhibits a considerable individual heterogeneity. Some individuals only had weak responses whereas others displayed strong platelet inhibition. The present flow cytometry technique appears suitable for identifying patients with abnormal reactions after clopidogrel exposure.
Angina Pectoris↗
ADP activation induces bFGF binding to platelets in vitro.
Basic fibroblast growth factor (bFGF), is a heparin-binding factor with potent angiogenic properties in vitro and in vivo. bFGF is involved in tumour growth, but it has also been shown to reduce infarct size in experimentally induced acute myocardial infarction. Platelets are also believed to have an important role in both tumour growth and myocardial infarction. We have studied bFGF binding to platelets by flow cytometry. Platelet activation by ADP induces bFGF binding to platelets. bFGF bound to activated platelets will result in a locally high concentration of bFGF in patients with myocardial infarctions and malignant tumours. Addition of recombinant bFGF to platelet rich plasma reduced the percentage of fibrinogen positive platelets. bFGF may thus have an inhibitory effect on platelet aggregation.
Adenosine Diphosphate↗
Factor V Leiden (G1691A) and prothrombin gene G20210A mutations as potential risk factors for venous thromboembolism after total hip or total knee replacement surgery.
Patients (n = 1600) from 12 European countries, scheduled for elective orthopaedic hip or knee surgery, were screened for Factor V Leiden and prothrombin gene G20210A mutations, found in 5.5% and 2.9% of the populations, respectively. All patients underwent prophylactic treatment with one of four doses of melagatran and ximelagatran or dalteparin, starting pre-operatively. Bilateral ascending venography was performed on study day 8-11. The patients were subsequently treated according to local routines and followed for 4-6 weeks postoperatively. The composite endpoint of screened deep vein thrombosis (DVT) and symptomatic pulmonary embolism (PE) during prophylaxis did not differ significantly between patients with or without these mutations. Symptomatic venous thromboembolism (VTE) during prophylaxis and follow-up (1.9%) was significantly over-represented among patients with the prothrombin gene G20210A mutation (p = 0.0002). A tendency towards increased risk of VTE was found with the Factor V Leiden mutation (p = 0.09). PE were few, but significantly over-represented in both the Factor V Leiden and prothrombin gene G20210A mutated patients (p = 0.03 and p = 0.05, respectively). However, since 90% of the patients with these genetic risk factors will not suffer a VTE event, a general pre-operative genotyping is, in our opinion, of questionable value.
Activated Protein C Resistance↗
Effect of melagatran on prothrombin time assays depends on the sensitivity of the thromboplastin and the final dilution of the plasma sample.
Prothrombin time (PT) assays are clotting methods that measure the activity of vitamin K-dependent coagulation factors (F) II, VII, and X. There are three main types of PT assays in general usage, namely the Quick assay, Owren's assay and PT dry chemistry test cards. PT assays were initially developed to monitor dose-adjustments of vitamin K antagonists such as warfarin. The aim of the present study was to investigate whether commercially available PT assays are suitable for evaluating the anticoagulant activity of direct thrombin inhibitors. Melagatran, a reversible direct thrombin inhibitor, was added to human plasma at concentrations ranging from 0.1 to 2.0 micromol/l. Seventeen different commercially available PT kits were used, including thirteen Quick reagents, two Owren reagents and two PT test cards. The sensitivity of the different reagents, expressed as the concentration of melagatran that doubled the prothrombin time (IC50) varied widely, with Thromboplastin S and Thromboplastin HS being the most sensitive (IC50 = 0.9 micromol/l). The reagents with apparently the lowest sensitivity were the two Owren reagents Nycotest PT and SPA 50 with an IC50 of 2.2 and 2.9 micromol/L, respectively. This is most likely due to a higher dilution of melagatran in these assays compared to the dilution in the Quick assays. The results were also dependent on the International Sensitivity Index (ISI) of each reagent. The concentration of melagatran that produced an International Normalized Ratio (INR) of 2 was calculated from dose-response curves for each assay, and these results revealed that reagents with a high ISI value gave an INR of 2 at much lower concentrations of melagatran (0.5-0.7 micromol/L) than those with an ISI-values around one (0.9-1.2 micromol/L). It was found that INR depends not only on the plasma concentration of melagatran, but also on the sensitivity of the PT reagent and on the final dilution of the plasma sample in the prothrombin time assay. Thus, since the same melagatran concentration can be associated with widely varying PT/INR results depending on the specific assay used it is concluded that PT assays and INR can not be used to monitor melagatran activity.
Anticoagulants↗
Acute effects of nicotine infusion on platelets in nicotine users with normal and impaired renal function.
The role of platelets in cardiovascular disease associated with smoking is becoming more established, but the effects of nicotine on platelets are unclear. Nicotine therapy is used for smoking cessation in both health and disease. Consequently, the effects of nicotine on platelets are of particular significance in disorders such as renal disease, which is associated with defective platelet function, increased cardiovascular morbidity, and altered nicotine metabolism. Thus, the aim of the present study was to investigate the acute effects of nicotine infusion (NI) on platelets in seven healthy subjects (HS) and seven patients with renal failure (RF). All subjects were nicotine users and had refrained from using nicotine for 36 h before NI. Blood was collected before, immediately after, and 2 h after NI. The plasma concentrations of nicotine and its main metabolite cotinine were determined by gas chromatography. Platelet responsiveness was assessed by aggregometry and flow cytometry in whole blood (P-selectin surface expression, fibrinogen- and von Willebrand factor-binding), P-selectin expression in isolated platelets, and immunoassays of platelet release (beta-thromboglobulin, platelet factor 4, and soluble P-selectin) and nitric oxide (NO) products. The plasma levels of cotinine, but not nicotine, were significantly higher in RF compared to HS at all time points. In both groups, collagen-induced platelet aggregation was restrained immediately after NI, when the plasma concentration of nicotine was maximal, and was restored after 2 h. Two hours after NI, activation-dependent P-selectin surface expression in isolated platelets increased in both groups. This increased platelet responsiveness occurred simultaneously with a significant increase of plasma cotinine and a decrease of NO products. Thus, the present study suggests that nicotine, directly or through some secondary mechanism or metabolite, only slightly potentiates some of the platelet responses. Renal failure appears not to influence the effects of nicotine on platelets.
Blood Platelets↗
Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation.
The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.
Animals↗
The use of platelet density and volume measurements to estimate the severity of pre-eclampsia.
BACKGROUND: This study evaluated whether it is possible to estimate the severity of pre-eclampsia through in vitro measurements of platelet and granulocyte parameters. EXPERIMENTAL PROTOCOL: Eighteen pre-eclamptic women in the third-trimester of pregnancy and 11 women in the third-trimester of normal pregnancies were included in the study. Three to 12 months after delivery, 15 patients with pre-eclampsia and all the subjects with normal pregnancies were re-examined. Before delivery, peak platelet density was determined using a specially designed apparatus. Before and 3-12 months after delivery the following were measured: platelet counts, mean platelet volume and neutrophil and monocyte counts. Furthermore, circulating P-selectin, interleukin-6 and myeloperoxidase were determined to estimate platelet, monocyte and granulocyte activities, respectively. RESULTS: Compared to their results after delivery, pre-eclamptic females demonstrated lower platelet counts (P < 0.001) and raised mean platelet volumes (P < 0.01). Both pre-eclamptic women (P < 0.01) and normal pregnancies (P < 0.05) demonstrated elevated soluble P-selectin at pregnancy. Then pre-eclamptic women had advanced neutrophil counts (P < 0.01) but normal pregnancies showed a similar phenomenon (P < 0.001). Interleukin-6 remained normal during pregnancy. Plasma myeloperoxidase levels were lower both in pre-eclampsia (P < 0.05) and in normal pregnancies (P < 0.001). In pre-eclampsia elevated blood pressure was related to higher mean platelet volumes (P < 0.05). Furthermore, a group of pre-eclamptic females whose platelets had disturbed density distribution displayed elevated mean platelet volumes (P < 0.01). CONCLUSIONS: The present work demonstrates considerable platelet alterations in pre-eclampsia. We failed to show granulocyte involvement in the pathogenesis of the disease. Severe pre-eclampsia is related to elevated mean platelet volumes. The latter parameter is associated with disturbed density distribution. It appears possible to estimate disease severity from measurements of platelet density and volume.
Adolescent↗
Blood plasma coagulation studied by surface plasmon resonance.
A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real time as a function of thromboplastin and heparin concentrations. The response curves were analyzed by curve fitting to a sigmoid curve equation, followed by extraction of the time constant. Clotting activation by thromboplastin resulted in increased time constant, as compared to spontaneously clotted plasma, in a dose dependent way. Addition of heparin to the thromboplastin-activated plasma counteracted this effect. Atomic force microscopy (AFM) pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and the fiber thickness increased with decreased thromboplastin concentration. The physical reason for the SPR signal observed is ambiguous and is therefore discussed. However, the results summarized in the plots and the fibrin network properties observed by AFM correlate well with present common methods used to analyze blood coagulation.
Anticoagulants↗
Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay.
It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.
Anticoagulants↗
A flow cytometric assay for the study of dense granule storage and release in human platelets.
The clinical manifestations of platelet dense ( delta ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83+/-6 (mean +/- 1 SD, range 69-91). The difference in MFI between resting and stimulated platelets was 28+/-7 (range 17-40). Six members of a family, of whom one had a known delta -storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval.
Journal Article↗
Laboratory diagnosis of thrombophilia by endothelial-cell-modulated coagulation.
This study investigated whether the addition of endothelial cells to blood or blood plasma is of value in global laboratory diagnostic testing for thrombotic tendency. Plasma from thrombotic patients and healthy individuals was exposed to human umbilical vein endothelial cells (HUVEC), in monolayers or suspensions, and fibrin deposition or clotting time, respectively, was registered. The latter was determined by a novel rheometric procedure that also gave information about coagulum rigidity. Plasma from patients (n = 10) tended to deposit more fibrin on HUVEC monolayers than plasma from healthy individuals (n = 10). When mixed with suspended HUVEC, plasma from patients (n = 14) showed shorter clotting times than plasma from healthy individuals [n = 13; 4.79 +/- 1.02 min (mean +/- SD) compared with 6.80 +/- 1.50 min, P < 0.001]. Coagulum rigidity among patients also differed from that of healthy individuals (P < 0.05). The study showed that the addition of endothelial cells to blood plasma is of value in global laboratory diagnostic testing for thrombotic tendency.
Adult↗