Neural foundation of the diathesis-stress model: longitudinal gray matter volume changes in response to stressful life events in major depressive disorder and healthy controls.

Recurrences of depressive episodes in major depressive disorder (MDD) can be explained by the diathesis-stress model, suggesting that stressful life events (SLEs) can trigger MDD episodes in individuals with pre-existing vulnerabilities. However, the longitudinal neurobiological impact of SLEs on gray matter volume (GMV) in MDD and its interaction with early-life adversity remains unresolved. In 754 participants aged 18-65 years (362 MDD patients; 392 healthy controls; HCs), we assessed longitudinal associations between SLEs (Life Events Questionnaire) and whole-brain GMV changes (3 Tesla MRI) during a 2-year interval, using voxel-based morphometry in SPM12/CAT12. We also explored the potential moderating role of childhood maltreatment (Childhood Trauma Questionnaire) on these associations. Over the 2-year interval, HCs demonstrated significant GMV reductions in the middle frontal, precentral, and postcentral gyri in response to higher levels of SLEs, while MDD patients showed no such GMV changes. Childhood maltreatment did not moderate these associations in either group. However, MDD patients who had at least one depressive episode during the 2-year interval, compared to those who did not, or HCs, showed GMV increases in the middle frontal, precentral, and postcentral gyri associated with an increase in SLEs and childhood maltreatment. Our findings indicate distinct GMV changes in response to SLEs between MDD patients and HCs. GMV decreases in HCs may represent adaptive responses to stress, whereas GMV increases in MDD patients with both childhood maltreatment and a depressive episode during the 2-year interval may indicate maladaptive changes, suggesting a neural foundation for the diathesis-stress model in MDD recurrences.

Changes in the degree of substitution of HES in vivo and their influence on methods for determining HES concentrations in plasma.

OBJECTIVE: The degree of substitution (DS) of HES describes the average proportion of substituted glucose molecules in a starch molecule. Although no quantitative studies of the in vivo behavior of the DS have been conducted so far, most pharmacokinetic studies to date have measured HES concentrations using the enzymatic method. This method assumes that at any point in time after an infusion, the DS in a serum remains constant and is identical to the DS in the infused solution. In the present study, we examined the changes in the DS of HES 130/0.42 in vivo in an animal model and compared two methods of measuring HES concentrations in plasma (the enzymatic and the o-Toluidine method). METHODOLOGY: We randomized 22 pigs into 2 groups. After induction of anesthesia, the pigs received 500 ml or 1000 ml of HES 130/0.42 (Tetraspan®). The DS was measured directly after the infusion, then after 30, 60, 120 and 240 minutes. In determining the DS, the hydroxyethyl starch was extracted from the plasma and hydrolyzed with hydrochloric acid to form non-substituted glucose and hydroxyethyl glucose. Subsequently, the concentration of free unsubstituted glucose was determined enzymatically and the total concentration of all (i.e., substituted and unsubstituted) glucose molecules was determined using the o-Toluidine method. From this, the concentration of the substituted glucose (hydroxyethyl glucose) and the DS could be calculated. In addition, the HES concentration was measured first in vitro and then in vivo at any point after the infusion by both the enzymatic method and the o-Toluidine method. RESULTS: The DS increased significantly directly after the infusion from 0.42 to 0.53 (for 500ml) or to 0.50 (for 1000ml); 4 hours later this had further increased to 0.55 and 0.54, respectively (p <0.0001). The HES concentration in vitro showed no significant difference (p = 0.17) when determined with the enzymatic and the o-Toluidine method. In contrast, the serum concentrations in vivo displayed significant differences (p<0.0001) between the two measurement methods. Immediately after the infusion of 500ml HES, the concentration measured with the o-Toluidine method was 31% higher than the one measured with the enzymatic method; 4 hours later, this discrepancy was still at 25%. For 1000 ml HES, the differences amounted to 16% and 25%, respectively. CONCLUSION: The DS of HES in vivo increases significantly over time. As a result, an HES concentration measured with the enzymatic method in vivo will be significantly lower than the same concentration determined with the o-Toluidine method. In future pharmacokinetic studies, HES concentrations should be measured using a method that takes into account changes in the DS in vivo.

Long-term predictive value of highly sensitive thyroglobulin measurement.

OBJECTIVE: To examine the predictive value of unremarkable nonstimulated highly sensitive thyroglobulin (hsTg) measurement with regard to the results of stimulated thyroglobulin (Tg) measurement, diagnostic whole-body scintigraphy, recurrence and differentiated thyroid cancer (DTC)-related death. DESIGN, PATIENTS AND MEASUREMENTS: We retrospectively analysed the data of all 461 (410 without anti-Tg-antibodies [TgAbs], 51 with) DTC patients who were referred to our department for treatment and follow-up care of differentiated thyroid cancer from 2004 onwards, and in whom at least one posttreatment Tg value was measured in our hospital at least 3 months after I-131 ablation. RESULTS: In the group of TgAb-negative patients, 2.0% of patients with an unstimulated Tg < 0.1 ng/ml showed a stimulated Tg ≥ 1.0 ng/ml, whereas this happened in 77.6% with an unstimulated Tg ≥ 0.1 but <1.0 ng/ml. An unstimulated hsTg ≥ 0.1 ng/ml had a sensitivity specificity positive and negative predictive value of 90.0%, 94.1%, 77.6% and 97.6%, respectively, for a stimulated Tg ≥ 1.0 ng/ml. In TgAb-positive patients, this was 75%, 97%, 75% and 97%, respectively. An unstimulated Tg ≥ 0.1 ng/ml did not significantly discriminate with regard to the risk of DTC-related death (p = .06), but ≥1.0 ng/ml did (p = .012), as did a stimulated Tg ≥ 1.0 ng/ml (p = .029). Excluding patients with distant metastases at diagnosis nullifies this significance. CONCLUSION: Except for patients with distant metastases, both TgAb negative and TgAb positive patients with an undetectable nonstimulated hsTg measurement have a very good prognosis. The high net present value of unstimulated hsTg testing means that further diagnostic procedures can be omitted in such patients.

Multi-platform Affinity Proteomics Identify Proteins Linked to Metastasis and Immune Suppression in Ovarian Cancer Plasma.

A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of proteins that are upregulated in the plasma of OC patients. Using binders targeting 368 different plasma proteins, we compared 20 plasma samples from HGSC patients (OC-plasma) with 20 plasma samples from individuals with non-malignant gynecologic disorders (N-plasma). We identified 176 proteins with significantly higher levels in OC-plasma compared to N-plasma by PEA (p < 0.05 by U-test; Benjamini-Hochberg corrected), which are mainly implicated in immune regulation and metastasis-associated processes, such as matrix remodeling, adhesion, migration and proliferation. A number of these proteins have not been reported in previous studies, such as BCAM, CDH6, DDR1, N2DL-2 (ULBP2), SPINT2, and WISP-1 (CCN4). Of these SPINT2, a protease inhibitor mainly derived from tumor cells within the HGSC microenvironment, showed the highest significance (p < 2 × 10-7) similar to the previously described IL-6 and PVRL4 (NECTIN4) proteins. Results were validated by means of the aptamer-based 1.3 k SOMAscan proteomic platform, which revealed a high inter-platform correlation with a median Spearman ρ of 0.62. Likewise, ELISA confirmed the PEA data for 10 out of 12 proteins analyzed, including SPINT2. These findings suggest that in contrast to other entities SPINT2 does not act as a tumor suppressor in HGSC. This is supported by data from the PRECOG and KM-Plotter meta-analysis databases, which point to a tumor-type-specific inverse association of SPINT2 gene expression with survival. Our data also demonstrate that both the PEA and SOMAscan affinity proteomics platforms bear considerable potential for the unbiased discovery of novel disease-associated biomarkers.

A new insole measurement system to detect bending and torsional moments at the human foot during footwear condition: a technical report.

BACKGROUND: Stress occurring at the feet while wearing footwear is often determined using pressure measurement systems. However, other forms of stress, such as bending, torsional and shear loadings, cannot be detected in shoes during day-to-day activities. Nevertheless, the detection of these types of stresses would be helpful for understanding the mechanical aspects of various kinds of hard and soft tissue injuries. Therefore, we describe the development of a new measuring device that allows the reliable determination of bending and torsional load at the foot in shoes. METHODS: The system consists of a measuring insole and an analogue device with Bluetooth interface. The specific shape of the insole base layer, the positions of the strain gauges, and the interconnections between them have all been selected in such a way so as to isolate bending and torsional moment detections in the medial and lateral metatarsal region. The system was calibrated using a classical two-point test procedure. A single case study was executed to evaluate the new device for practical use. This application consisted of one subject wearing neutral shoes walking on a treadmill. RESULTS: The calibration results (coefficients of determination R(2) > 0.999) show that bending and torsional load can be reliably detected using the measurement system presented. In the single case study, alternating bending and torsional load can be detected during walking, and the shape of the detected bending moments can be confirmed by the measurements of Arndt et al. (J Biomech 35:621-8, 2002). CONCLUSIONS: Despite some limitations, the presented device allows for the reliable determination of bending and torsional stresses at the foot in shoes.

Intravitreal functional plasminogen in eyes with branch retinal vein occlusion.

PURPOSE: To evaluate whether intravitreal functional plasminogen is elevated in eyes with branch retinal vein occlusion (BRVO) and to discover whether intravitreal plasminogen activities are correlated with the extent of blood-retina barrier (BRB) breakdown. METHODS: Our study is a prospective case series of 20 consecutive patients with BRVO and 10 consecutive patients serving as controls. Vitreous taps were extracted from the central vitreous body and plasminogen was functionally determined in an innovative, ultrasensitive p-nitroanilide reaction after activation with streptokinase (100% of normal, %N = functional plasminogen in pooled normal citrated plasma). Intravitreal VEGF levels were assayed to estimate BRB breakdown. RESULTS: Intravitreal functional plasminogen was detected in all analyzed samples (n = 30) and mean (±SD) plasminogen activities were found to be 0.97 ± 1.06%N (range: 0.03-3.9%N). Patients suffering from BRVO exhibited significantly higher intravitreal plasminogen (1.35 ± 1.11%N) in comparison with controls (0.20 ± 0.21%N, p < 0.001). Intravitreal VEGF concentrations in the BRVO group (576 ± 547 pg/ml) were significantly higher than these in controls (111 ± 120 pg/ml, p = 0.003). There was a significant correlation between intravitreal functional plasminogen and intravitreal VEGF levels (r = 0.519, p = 0.003). CONCLUSIONS: Intravitreal functional plasminogen is significantly elevated in eyes suffering from BRVO and correlates with the extent of BRB breakdown. The induction of posterior vitreous detachment by using intravitreally administered recombinant tissue plasminogen activator (enzymatic vitreolysis) should be explored in further investigations.

Soluble opsin is present in human vitreous.

The purpose of this research project was to evaluate if intravitreal opsins are present in human vitreous liquid which is, so far, unknown. Therefore a pilot study was conducted including 22 vitreal samples which were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy for macular pucker, diabetic vitreous hemorrhage or vitreal floater removal as well as macular hole closure or vitreomacular traction relief from the central vitreous body. No adverse events or serious side effects occurred. All samples were immediately stabilized by human albumin and arginine and subsequently frozen. Short-wavelength cone opsin concentrations were analyzed by enzyme immune essay (EIA) with anti-proteolytic 400 mM arginine, pH 8.7, in the antigen capture phase. Intravitreal short-wavelength cone opsins were detected in all analyzed samples and respective concentrations ranged at levels of 157 pg/ml ± 73 pg/ml (MV ± SD; range: 27 pg/m-286 pg/ml). Eyes with MP/MH/DVH/VMT and VF exhibited intravitreal short-wavelength cone opsin concentrations of 189 pg/ml ± 68 pg/ml (range: 72 pg/ml-286 pg/ml)/96 pg/ml ± 39 pg/ml (range: 50 pg/ml-138 pg/ml)/126 pg/ml ± 88 pg/ml (range: 27 pg/ml-198 pg/ml)/224 pg/ml and 121 pg/ml. Further studies will quantify the intravitreal opsin pattern of all visual opsins and compare these concentrations between different vitreoretinal diseases. This in turn might offer a better pathophysiological understanding and new diagnostic and therapeutic strategies for various eye pathologies. As a hypothesis, soluble opsins might be a biomarker for retinal damage comparable to creatinine for kidney damage.

Intravitreal thrombin activity is elevated in retinal vein occlusion.

To evaluate whether intravitreal thrombin activity is elevated in eyes with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) in comparison to healthy controls. Prospective clinical case series of 19 patients with BRVO, 13 patients suffering from CRVO and nine participants serving as controls. Vitreous taps were extracted from the central vitreous body, 200 µl frozen/thawed sample was immediately stabilized with 200 µl 5% human albumin, and 200 µl mixture thereof was stabilized with 200 µl 2.5 mol/l arginine, pH 8.6. Thrombin activity was determined chromogenically. Intravitreal levels of vascular endothelial growth factor (VEGF) as a marker for blood-retina barrier (BRB) breakdown were measured by a commercial chemiluminescent enzyme immuno assay (R&D). Intravitreal thrombin activity and VEGF levels were 1.6 ±â€Š1.2 mIU/ml (mean value ±â€ŠSD; range: 0.2-4.2 mIU/ml) and 554 ±â€Š568 pg/ml (range: 20-2005 pg/ml) in BRVO-affected eyes, 2.6 ±â€Š1.2 mIU/ml (range: 0.8-5.2 mIU/ml) and 1332 ±â€Š1350 pg/ml (range: 58-3943 pg/ml) in eyes suffering from CRVO as well as 0.8 ±â€Š0.8 mIU/ml (range: 0.2-2.7 mIU/ml) and 115 ±â€Š120 pg/ml (range: 32-431 pg/ml) in controls. There are significant differences of intravitreal thrombin activity and intravitreal VEGF levels between eyes with BRVO, CRVO, and controls (P = 0.007 and P = 0.003, Kruskal-Wallis test). Intravitreal thrombin activity is significantly correlated with intravitreal VEGF levels (r = 0656; P < 0.001, Pearson correlation). Intravitreal thrombin activity might serve as a new marker for BRB breakdown or macular fibrin deposition in ophthalmology. Significant differences of intravitreal thrombin activity between eyes with BRVO, CRVO, and healthy controls might offer new therapeutic strategies for RVO-affected eyes. The effect of oral and intravitrealy injected direct thrombin inhibitors needs to be evaluated in further investigations.

Intravitreal vascular endothelial growth factor.

PURPOSE: To evaluate whether a specific pre-analytical stabilization regimen is needed for naïve vitreous taps to detect true values of intrinsic VEGF levels. METHODS: Fourteen consecutive patients with different vitreomacular pathologies without blood-retina-barrier breakdown were scheduled for standard 23-gauge three-port pars plana vitrectomy, and naïve vitreous taps were sampled at the beginning of each procedure. The extracted vitreous specimen was split; one half was immediately stored in a -20 °C freezer (unstabilized samples) and the other half was instantly stabilized with albumin (2.5 % final conc.), followed by arginine stabilization (1.25 M final conc.) and consecutively stored in a -20 °C freezer (stabilized samples). RESULTS: Intravitreal VEGF was detected in all 14 analyzed samples (100 %). VEGF levels were shown to be 46.5 pg/ml ± 62.3 pg/ml (MV ± SD; range: 5.99-232.3 pg/ml) in unstabilized, and 120.4 pg/ml ± 94.4 pg/ml (range: 42.9 pg/ml-289.6 pg/ml) in stabilized vitreous samples. Intravitreal VEGF levels in stabilized vitreous samples were on average 2.6-fold, and thus significantly higher than in unstabilized taps of same eyes (p = 0.001, Wilcoxon test). VEGF levels in stabilized vitreous samples can be up to 8.5 times higher than in corresponding unstabilized vitreous taps of same eyes (bootstrap analysis). Intravitreal VEGF levels in unstabilized samples correlate with those in stabilized vitreous taps (r = 0.594; p = 0.025; Pearson). CONCLUSIONS: An adequate pre-analytic stabilization regimen is needed to evaluate the most accurate intravitreal VEGF levels. This in turn will result in a better understanding of the physiological as well as pathological role of VEGF within the eye. Furthermore, knowing the true value of intravitreal VEGF levels will help to calculate the dosage of intravitrealy applied anti-VEGF drugs.

Fibrinolysis in normal vitreous liquid.

Plasmin is the key enzyme of fibrinolysis. Plasmin-alpha2-antiplasmin (PAP) complexes are biomarkers of fibrinolysis activation. The purpose of our investigation was to evaluate the activity of fibrinolysis in normal human eyes, that is in eyes without blood-retina barrier breakdown (BRB), which has not been investigated so far. Twenty-two vitreal samples were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy for macular pucker removal, macular hole closure or vitreal floater removal from the central vitreous body. These samples were immediately stabilized with human albumin (2.5% final conc.) and arginine (1.25 mmol/l final conc.) and subsequently frozen. Plasminogen was functionally determined in an ultra-sensitive pNA reaction after activation with streptokinase (100% = functional plasminogen in pooled normal citrated plasma). PAP concentrations were measured by enzyme immune assay (EIA). Intravitreal functional plasminogen exhibited to be 1 ±â€Š0.65% (range: 0.2-2.49%). PAP concentrations ranged at levels of 14 ±â€Š9ng/ml (range: 2-33 ng/ml). Pearson's correlation quotient between functional plasminogen and PAP revealed to be r equal to -0.27 (P = 0.221). No adverse events or serious side effects occurred. Sampling vitreous fluid at the beginning of a standard 23-gauge three-port pars plana vitrectomy is a well tolerated procedure. A strict stabilization procedure for extracted vitreous specimen is necessary to obtain activities and concentrations that are close to the true intraocular value. There is a basal intraocular fibrinolysis, a possible target for intravitreal pharmacological therapy.

Thrombin activity in normal vitreous liquid.

An intact blood-retina barrier (BRB) ensures the homeostatic regulation of the retinal environment by preventing proteins and enzymes to pass from the blood stream into the retinal tissue as well as the vitreous cavity ('nonleaky eyes'). Nevertheless, thrombin is needed within the eye to avoid bleeding events. It might, furthermore, play an essential role in preventing BRB breakdown ('leaky eyes'). Until today, the intraocular thrombin activity as well as the source of the latter has not been investigated. The present work was conducted to evaluate whether intravitreal thrombin activity is present in eyes without BRB breakdown. Therefore, 16 vitreal taps were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy. These 200 µl undiluted vitreous samples were instantly stabilized by 1 + 1 mixture with 5% human albumin, followed by 1 + 1 mixture of such an aliquot with 2.5 mol/l arginine, pH 8.6, and frozen at -20°C. After thawing at 23°C, thrombin activity was analyzed chromogenically in the presence of arginine protection against unspecific cleavage of the chromogenic substrate. Intravitreal thrombin was detected in all 16 analyzed samples and thrombin activity exhibited to be 1.5 ±â€Š1.0 mIU/ml (mean value ±â€ŠSD; range: 0.2-3.25 mIU/ml). Thus, our investigation is the first successful quantification of the physiologic intraocular activity of thrombin. Further studies will evaluate intravitreal thrombin activities in eyes with BRB breakdown and compare those results with the physiologic activities demonstrated herein. Standardized intraocular thrombin activity might be a new diagnostic parameter in ophthalmology.

Intravitreal functional plasminogen is elevated in central retinal vein occlusion.

PURPOSE: To detect intravitreal functional plasminogen in vitreous samples of patients with recent onset of central retinal vein occlusion (CRVO) and to demonstrate significantly higher intravitreal plasminogen in CRVO patients in comparison to controls. METHODS: Prospective clinical case series of 13 consecutive patients with recent onset of CRVO scheduled for core pars plana vitrectomy and 10 consecutive patients undergoing standard pars plana vitrectomy for routine macular surgery or vitreal floater removal. In all 23 cases, vitreous taps were extracted from the central vitreous body, and plasminogen was functionally determined in a new ultrasensitive p-nitroanilide reaction after activation with streptokinase (100% of normal, %N = functional plasminogen in pooled normal citrated plasma). RESULTS: Plasminogen was detected in all analyzed samples (n = 23), and mean plasminogen was revealed to be 1.33 ± 1.73% (mean ± SD), with a range of 0.03-7.8%N. Patients with recent onset of CRVO exhibited significantly higher intravitreal plasminogen (2.19 ± 1.89%N) in comparison to controls (0.20 ± 0.21%N; p < 0.001, Mann-Whitney U test). CONCLUSION: Due to significantly increased intravitreal plasminogen in patients with recent onset of CRVO, intravitreally administered tissue plasminogen activator might be an option to induce posterior vitreous detachment (enzymatic vitreolysis) in CRVO patients.

Diagnostic ultrasound activates pure prekallikrein.

Diagnostic ultrasound activates the contact phase of human coagulation. This has been seen in human blood or plasma or with purified factor 12. The present work aimed to quantify a possibly triggering action of ultrasound on purified prekallikrein, the second of the two main triggers of the intrinsic hemostasis cascade. Either 2.7 µg/ml human prekallikrein or for control 1 µg/ml kallikrein in 26% glycerol - 0.54% NaCl-10.6 mmol/l Na3 citrate pH 7.4, in emptied polypropylene coagulation monovettes (Sarstedt) were exposed to diagnostic ultrasound (Siemens Acouson Antares, 5 MHz, 0.6 TIB, 0.6 TIS) for 0-5 min at room temperature (RT). Fifty microliter samples were withdrawn in duplicate and placed into an U-wells high quality microtiter plate (Brand 781600). Then 10 µl 2 mmol/l chromogenic substrate HD-CHG-Ala-Arg-pNA in 0.45% NaCl were added, and the increase in absorbance with time (ΔA405 nm /t at 37°C) was determined by a microtiterplate photometer with a 1 mA resolution (PHOmo; anthos). Exposure to diagnostic ultrasound biphasically increased the chromogenic activity of a prekallikrein solution in 26% glycerol. About 3-4 min ultrasound at 23 °C generated about 0.02 µg/ml kallikrein, that means that about 1% of pure prekallikrein in glycerol was converted into kallikrein. Thus, diagnostic ultrasound activates purified human prekallikrein to kallikrein. The ultrasound energy seems to fold the latent proenzyme prekallikrein into the active enzyme kallikrein. This contributes to explain the triggering action of ultrasound on the contact system of plasmatic human coagulation. Conversion of only 1% of prekallikrein into kallikrein is absolutely sufficient to start the intrinsic coagulation cascade. The clinical consequence of this action of ultrasound on intrinsic coagulation is that patients at risk for thrombosis, for example, patients with insufficiencies of hepatocytes, AT-3, C1-ina, or fibrinolysis should be protected by low-molecular-weight-heparin prior to the exposure of ultrasound, especially upon its prolonged exposure.

Determination of the anti-F10a or anti-F2a generation action of rivaroxaban or dabigatran.

Dabigatran and rivaroxaban are new oral inhibitors of the thrombin (F2a) and F10a, respectively. Both inhibitors decrease the extrinsic or intrinsic generation of F10a/F2a in plasma. An innovative test for extrinsic F10a/F2a generation is the extrinsic coagulation activity assay (EXCA). With the EXCA the action of all F10a/F2a generation inhibitors can be quantified. The present work aimed to establish the mean therapeutic ranges of these two new drugs, as determined in the EXCA. Forty microlitres reconstituted lyophilized commercial pooled normal plasma, that had been supplemented with 0-0.48 mg/l dabigatran or rivaroxaban, in high-quality polystyrene U-wells (Brand781600), were incubated with 4 µl 1 ng/ml human placental tissue factor in 5% human albumin, 250 mmol/l CaCl2 (EXCA-trigger) for 1 min (EXCA-1) or 2 min (EXCA-2) at 37°C. 80 µl 2.5 mol/l arginine, 0.16% Triton X 100, pH 8.6, were added. After 3 min, 20 µl 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine was added and ΔA/t was determined (37°C). The mean therapeutic range (10-20% EXCA) of dabigatran is approximately 0.05-0.1 mg/l. The mean therapeutic range (10-20% EXCA) of rivaroxaban is approximately 0.13-0.22 mg/l. Mean prophylactic concentrations (20-40% EXCA) are approximately 0.02-0.05 mg/l dabigatran and approximately 0.05-0.13 mg/l rivaroxaban. These are the mean ranges. Individual plasma might well behave differently: either an individual patient could be more sensible or more resistant towards one or both of these drugs. It is suggested to perform EXCA tests for each individual patient to determine the real drug dosage he needs to reach 10-20% of normal EXCA for therapy or 20-40% of normal EXCA for prophylaxis.

The ultrasound frequency determines the degree of intrinsic coagulation activation.

Diagnostic ultrasound activates intrinsic coagulation. The aim of the present work was to quantify the action of different ultrasound frequencies on the contact phase of human blood coagulation. Pooled normal citrated platelet-poor plasma in 2 ml aliquots in polypropylene monovettes was exposed to diagnostic ultrasound, changing the ultrasound frequency from 17 to 15 to 12 to 8 to 7 MHz (at an intensity of 1.1 MI). After 0-2 min (23°C), 400 µl samples were withdrawn and placed into polypropylene Eppendorf cups. Forty microliters of plasma sample was pipetted into U-wells polystyrene microtiter plates of high purity (Brand781600). Immediately thereafter, the recalcified coagulation activity assay (RECA) was performed. Seventeen megahertz ultrasound exposure was the weakest activator of intrinsic coagulation of all frequencies tested: even 2 min of exposure at 23°C enhanced F2a generation by only about three-fold. The shorter the ultrasound exposure, the better the action against intrinsic hemostasis: 0.5 min of ultrasound exposure at 23°C induced less than two-fold thrombin generation in all frequencies tested. One minute ultrasound exposure (23°C) triggered intrinsic coagulation strongest at 8 MHz, showing an approximately four-fold increase in F2a generation. A 1.5 min of ultrasound exposure (23°C) triggered coagulation strongest at 7 MHz, showing an approximately 14-fold increase in F2a generation. Two minute of ultrasound exposure (23°C) triggered coagulation strongest at 15 MHz, showing an approximately 18-fold increase in F2a generation. Ultrasound has to be considered as a potential inducer of pathologic systemic coagulation. Patients at risk for increased coagulation activation and/or liver insufficiency should be protected with low molecular weight heparin, if a prolonged ultrasound diagnostic is planned. Ultrasound frequencies of about 17 MHz are the weakest activators of intrinsic coagulation. Ultrasound frequencies of about 7 MHz might be used for therapeutic induction of coagulation activation, such as in patients with severe cerebral or hepatic hemorrhages.

Contact activation of coagulation depends on the maximal lipophilic trigger concentration.

Contact activation of hemostasis is of great clinical importance. Intrinsic coagulation starts upon blood matrix change, resulting in activation of factor XII (F XII) to F XIIa and/or of prekallikrein to kallikrein. The intrinsic system is very complex. There are many reactions that can increase or decrease the generation of F Xa/thrombin. Currently, there are two main trigger types that activate the intrinsic system: (delta)-negatively charged molecules and lipophilic molecules. Recently, it was shown that the stimulation of thrombin generation by (delta)-negatively charged molecules depends on their maximal plasma concentration prior to plasma dilution. The questions arise whether this is also true for lipophilic triggers. Fifty-microliter frozen/thawed pooled normal platelet-poor citrated plasma (PNP) were supplemented with up to 5% (final concentration) hexane, followed by repetitive 1 + 1 dilutions on polystyrene U-wells microtiter plates of high quality (Brand, Wertheim, Germany; article number 781600). Immediately thereafter, the recalcified coagulation activity assay was started and the approximate 200% stimulatory concentrations (approx. SC200s) on intrinsic thrombin generation were determined, the 100% control being unsupplemented PNP. The higher the maximal concentration of hexane prior to dilution, the higher the approx. SC200. If the maximal hexane concentration prior to dilution was higher than 2%, PNP had an approx. SC200 of 0.1-0.2% (8-15 mmol/l) hexane. At maximal hexane concentrations prior to dilution in the range of 0.1-1%, the approx. SC200 decreased 10-fold to about 0.01-0.02%. Up to about 0.1% maximal hexane concentration prior to dilution the corresponding approx. SC200 of hexane on intrinsic thrombin generation was 0.003-0.01% (0.2-0.8 mmol/l). Both main types of contact triggers - negatively charged or lipophilic molecules - have a peculiar behavior respective to maximal plasma concentration/thrombin generation: if the maximal plasma concentration of the trigger prior to plasma dilution is high, then the thrombin generating system needs rather high amounts of triggers to reach approx. SC200; and, if the maximal plasma concentration of the trigger prior to dilution is low, then thrombin is easily generated with a low approx. SC200. This means that the plasmatic F XII/prekallikrein/HMWK system could be inhibited by high plasma concentrations of any trigger and this inhibition cannot be reversed by plasma dilution. To study the action of drugs on the intrinsic system of plasma, plasma should be supplemented with the respective drug at maximal concentrations that are in the range of the maximal blood concentrations obtained in clinical medicine.