Plasma Of Healthy Donors Research Articles

UP-2.047: Reactive Oxygen Species (ROS) Level in Seminal Plasma of Infertile Men and Healthy Donors

Reactive oxygen species (ROS) are a group of free radicals that in excessive amounts showed to have negative influence on sperm quality and function. We compared ROS levels in seminal plasma of infertile men with its level in healthy donors. We also determined the ROS level in semen of infertile patients according to the etiology of infertility, and also the effect of smoking on its level.

Oxidative stress and total antioxidant capacity in human plasma

While several tools are already available for the separate measurement of the oxidant and antioxidant pools, a single, quick and easy method for determining total oxidative stress would be advantageous. In the present study, we compare the plasma of untreated patients with leukemia/solid gynecological tumors (n = 50) and current regular smokers (n = 50) with a smoking history of ≥10 cigarettes per day to the plasma of healthy blood donors. Standard tools were used to measure total oxidant status, ceruloplasmin activity, total antioxidant capacity, uric acid content and oxidative stress index. Oxidative stress was also evaluated using the controversial d-ROMs test, a commercial method of reactive oxygen species detection. Statistically significant differences between the smokers and the control group were detected for all of the biochemical parameters. Conversely, the differences in the cancer patients were not statistically significant for oxidative stress.

Activity of clotting factors in fresh‐frozen plasma during storage at 4°C over 6 days

Fresh-frozen plasma (FFP) requires thawing, which delays availability. We investigated clotting factor activity and bacterial contamination of FFP when stored at 4 degrees C +/- 2 degrees C for 6 days. Plasma of 20 healthy plasma donors was sampled, frozen, and analyzed at baseline and repeatedly over a period of 6 days after thawing. The activity of fibrinogen, Factor (F)II, FV, FVII, FVIII, F IX, FX, XI, FXII, FXIII, antithrombin III (ATIII), von Willebrand factor antigen (VWF-Ag), protein C (PC), and free protein S (FPS) were determined and analyzed over time. Immediately after thawing there was a significant decrease of fibrinogen (-9%), FII (-7%), FV (-14%), FVII (-12%), FX (-11%), FXIII (-20%), PC (-7%), and ATIII (-4%), whereas FVIII (+8%), F IX (+1%), FXI (+11%), FXII (-1%), FPS (-1%), and VWF-Ag (-6%) remained stable without significant change. Over 6 days after thawing fibrinogen, ATIII (+2%) and VWF-Ag (+2%) remained stable whereas FXII (+2%), FXIII (+6%), and PC (+3%) changed significantly over time and increased at the end. FII (-8%), FV (-16%), FVII (-31%), FVIII (-47%), F IX (-12%), FX (-10%), FXI (-25%), and FPS (+/-0%) changed also significantly over time and decreased at the end. All clotting factors and inhibitors remained within the reference range requested by quality assurance regulations. No FFP bag showed bacterial contamination. This provides evidence for maintaining quality of thawed FFP and may improve rapid availability in emergency situations and reduce cost for health care givers.

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion.

Production of reactive oxygen species by monocyte-derived macrophages from blood of healthy donors and patients with ischemic heart disease

Production of reactive oxygen species (ROS) by macrophages derived from blood monocytes of healthy donors (MPN) and patients with ischemic heart disease (IHD) (MPIHD) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from blood plasma of healthy donors (LDLN) and patients with a high cholesterol level (LDLH) was investigated by the method of luminol-dependent (spontaneous) and stimulated chemiluminescence (CL) using opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) as the CL stimulators. It was shown that proper, luminol-dependent, and zymosan-or PMA-stimulated chemiluminescence of MPIHD was 1.4-, 1.8-, 2.7-, and 1.6-fold higher than the same types of chemiluminescence of MPN, respectively, (p<0.05–0.01). Although the effect of OZ on MPN and MPIHD was more potent than that of PMA (by 4.3- and 3.2-fold, respectively), but it appeared in 2.5–3.0 times slower than that of PMA. LDLN and LDLH incubated with MPN for the first 15 and 60 min caused the 1.4- and 2.5-increase of the luminol-dependent CL of MPN; the same treatment of MPIHD did not influence ROS production by these cells. Repeated increase in the OZ-stimulated CL of MPN was also observed after preincubation for 15–180 min with LDLN and LDLH followed by LDL removal, subsequent MPN washing and addition of Hanks solution and OZ; the repeated increase in OZ-stimulated CL of MPN was only observed after incubation with LDLH than with LDLN. No increase of CL was observed in experiments with MPIHD. Thus, more intensive chemiluminescence of macrophages obtained from blood of patients with IHD suggests their in vivo stimulation. LDLN and LDLH may cause both primary and secondary (after preincubation) stimulating effect on CL of MPN but not of MPIHD. Thus, the analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage stimulation; it may be effectively used for monitoring of effectiveness of medical treatment of patients.

Comparison of the Abbott RealTime Human Immunodeficiency Virus Type 1 (HIV-1) Assay to the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test: Workflow, Reliability, and Direct Costs

The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay (ART) and the Cobas AmpliPrep/Cobas TaqMan HIV-1 test (CTM) are commercially available assays for quantification of HIV-1 RNA in plasma. We evaluated performance characteristics, workflow, throughput, reliability, and direct costs of these assays. Both assays yielded good correlation of quantitative results (r = 0.95) among clinical specimens, with a mean difference of -0.34 log(10) copies/ml. Testing of healthy donor plasma specimens yielded "target not detected" results by ART, with "HIV-1 RNA detected, <40 copies/ml" results for 3.3% (3 of 90 samples) of these specimens by CTM. Both the m2000sp/m2000rt (ART) and docked CAP/CTM96 (CTM) instrument systems were capable of operating with continuous, uninterrupted workflow. When daily maintenance and cleaning were included, ART and CTM run durations (5 h 52 min and 6 h 4 min, respectively) and hands-on times (53 min and 46 min, respectively) were similar for a run batch size of 24. While ART was more flexible in terms of run batch size, CTM required fewer user interventions and consistently produced higher specimen throughput rates at 8, 16, and 24 h. Assay run failure rates were 6.3% (1 of 16 runs) and 4.2% (1 of 24 runs) for ART and CTM, respectively (P = 1.000), with invalid specimen result rates of 1.0% (5 of 495 specimens) and 2.8% (11 of 399 specimens), respectively (P = 0.073). Direct reagent and consumable costs for each assay were comparable (difference of <10%). In selecting an assay for implementation, laboratories should consider how various assay and instrument features might impact laboratory operation and patient care.

Lectin Activity of the Coagulation Factor VIII/von Willebrand Complex

The human coagulation factor VIII (FVIII) is essential in the intrinsic pathway of blood coagulation and circulates mainly as a non-covalently bound complex with the von Willebrand factor (VWF). This complex (FVIII/VWF) protects FVIII from degradation and cellular uptake, although no biological role has been identified yet for this complex. The FVIII/VWF complex was purified from a healthy donor's plasma by affinity chromatography on a Sepharose 4B-Concanavalin A column and was used to determine its capability to interact with erythrocytes and platelets. The purified FVIII/VWF complex at 6.0 and 12 microg/ml agglutinates rabbit and bovine erythrocytes, and showed negative agglutination with erythrocytes from other species including human ABO. Treatment of erythrocytes with Clostridium perfringens sialidase or trypsin increased four-fold the activity toward rabbit erythrocytes and positive agglutination for human A and B erythrocytes, suggesting the presence of FVIII/VWF-cryptic receptors in these erythrocytes. Goat, pig, or human O erythrocytes were not agglutinated even after enzymatic treatment. Fucose or N-acetyl-glucosamine (GlcNAc), at 10 mM, inhibited agglutinating activity of the complex with rabbit, human A and B erythrocytes, whereas galactose and N-acetyl-galactosamine, even at 200 mM, showed no effect on the complex activity. The FVIII/VWF complex, at 1.5 microg/200,000 platelets, significantly decreased platelet aggregation (p < 0.001) when compared with the effect of platelet-rich plasma; this effect was inhibited with 15 mM GlcNAc or fucose. ELISA assays on FVIII/VWF coated polystyrene plates confirmed specific binding to fucose- or biotinylated GlcNAc-dextran derivatives. We therefore propose that the FVIII/VWF complex possesses lectin activity.

Anti-interferon-γ autoantibody in a patient with disseminated Mycobacterium avium complex

We report the case of a 44-year-old woman with disseminated Mycobacterium avium complex (MAC) infection involving multiple bone lesions despite a normal healthy status until 6 months previously. Because she was suspected to have acquired immunodeficiency, we tested interferon (IFN)-gamma production by peripheral blood mononuclear cells (PBMC) after phytohemagglutinin (PHA) or anti-CD3 stimulation, and found that these cells produced no, or undetectable, levels of IFN-gamma in the presence of the patient's plasma, but produced nearly normal levels of IFN-gamma in the presence of healthy donor plasma. Since the IgG fraction of the patient's plasma was capable of blocking in vitro responses to IFN-gamma, the cause of disseminated MAC infection in this case appeared to be anti-IFN-gamma autoantibodies. To reduce the titer of anti-IFN-gamma autoantibodies, the patient received intravenous immunoglobulin (IVIG). However, titer of autoantibodies changed little compared to that before IVIG administration. According to our literature search, this is only the second case of disseminated MAC infection associated with anti-IFN-gamma autoantibodies in Japan.

Increased Plasma Concentrations of Cytoskeletal and Ca2+-Binding Proteins and Their Peptides in Psoriasis Patients

The mechanisms underlying psoriatic pathogenesis are not fully understood and might be elucidated by identifying novel disease-related molecular markers, including autoantigens. We used 2 proteomic methods to analyze plasma samples from 20 psoriasis patients and 20 matched healthy donors. The first method focused on evaluating changes in glycoprotein concentrations and the plasma proteome, and the second method assessed endogenous proteolytic activity by analyzing the low molecular weight component of plasma. The integrated proteomic and peptidomic analysis identified a number of proteins and their fragments present at different concentrations in the plasma of psoriasis patients and healthy donors. We used ELISA to independently verify the changes in the concentrations of several of these proteins. One intriguing finding, increased concentrations of cytoskeletal and actin-binding proteins and their peptides in psoriatic plasma, suggested disease-related cell leakage of these proteins and their increased proteolysis. Among the increased proteins and peptides were thymosin beta 4, talin 1, actin gamma, filamin, and profilin. Increased concentrations of Ca(2+)-binding proteins calgranulins A and B in psoriatic plasma were also observed, confirming previous reports, and appeared to be relevant to the increase of cytoskeletal components. Another notable change in psoriatic plasma was a striking decrease in fibrinogen fragments. The identified increased concentrations of cytoskeletal proteins, their peptides, and calgranulins in psoriatic plasma, as well as the underlying altered protease activity, are proposed to be related to psoriasis pathogenesis.

Banking of Biological Fluids for Studies of Disease-associated Protein Biomarkers

With the increasing demand of providing personalized medicine the need for biobanking of biological material from individual patients has increased. Such samples are essential for molecular research aimed at characterizing diseases at several levels ranging from epidemiology and diagnostic and prognostic classification to prediction of response to therapy. Clinically validated biomarkers may provide information to be used for diagnosis, screening, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease), and prediction of response to treatment and as a surrogate response marker. Many types of biological fluids or tissues can be collected and stored in biorepositories. Samples of blood can be further processed into plasma and serum, and tissue pieces can be either frozen or fixed in formalin and then embedded into paraffin. The present review focuses on biological fluids, especially serum and plasma, intended for study of protein biomarkers. In biomarker studies the process from the decision to take a sample from an individual to the moment the sample is safely placed in the biobank consists of several phases including collection of samples, transport of the samples, and handling and storage of samples. Critical points in each step important for high quality biomarker studies are described in this review. Failure to develop and adhere to robust standardized protocols may have significant consequences as the quality of the material stored in the biobank as well as conclusions and clinical recommendations based on analysis of such material may be severely affected.

Constitutive expression of the antibacterial CXC chemokine GCP-2/CXCL6 by epithelial cells of the male reproductive tract

The reproductive tract is continuously challenged by potential pathogens present in the environment. Therefore, robust host defense mechanisms are essential both for the health of the individual and for fertilization. Antibiotic innate immunity peptides possess broad antimicrobial activity. Recently, we found that the CXC chemokine, granulocyte chemotactic protein (GCP)-2/CXCL6, possesses antibacterial activity. In the present study, we investigated, therefore, the presence of GCP-2/CXCL6 in the human male reproductive system. GCP-2/CXCL6 was detected at 19 nM (mean; range: 5–47 nM; n = 14) in seminal plasma of fertile donors, i.e. at levels more than 100 times higher than those previously reported for the related chemokine IL-8/CXCL8. No GCP-2/CXCL6 could be detected in blood plasma of healthy donors, indicating local production in the male reproductive tract. In vasectomized donors, significantly lower levels of GCP-2/CXCL6 were found (mean: 3 nM; range 2–7 nM; n = 7), demonstrating that the testis and epididymis contribute significantly to the GCP-2/CXCL6 content of seminal plasma. Strong expression of GCP-2/CXCL6 was found in the epithelium of the testis, epididymis and seminal vesicles, while the prostate epithelium showed weak expression, as determined by immunohistochemistry. A biological function is suggested, viz. at concentrations of the order of those found in seminal plasma, GCP-2/CXCL6 has antibacterial activity against the urogenital pathogen Neisseria gonorrhoeae. GCP-2/CXCL6 in seminal plasma may play roles in both host defense of the male urogenital tract and during fertilization.

Assessment of circulating proteasome chymotrypsin-like activity in plasma of patients with acute and chronic leukemias

Objective We evaluated whether the proteasomal chymotrypsin-like (ChT-L) activity is increased in plasma of patients with acute lymphoblastic (ALL), acute myeloblastic (AML) and chronic lymphocytic (CLL) leukemias. Methods The activity was assayed using the fluorogenic peptide substrate in the presence of an artificial activator sodium dodecyl sulfate (SDS) in the plasma of healthy donors ( n = 15) and ALL ( n = 15), AML ( n = 28) and CLL ( n = 22) patients. Results The activity was significantly ( P < 0.001) higher in the plasma of ALL and AML patients at the diagnosis than in healthy subjects and decreased after therapy or remained unchanged or rose during relapse. By contrast, in CLL patients at the diagnosis, the activity did not differ significantly from the healthy controls. In each group, the activity positively correlated with the serum lactic dehydrogenase activity. Conclusions Plasma proteasome ChT-L activity can be a useful bio-marker for patients with acute leukemia at the blast stage.

Changes in amino acid patterns of blood plasma in tumor patients with the anticancer drug NSC-631570: Possible approaches to cancer diagnostics

In our early experimental (with W-256, SM-1 and PC-1 tumors) and clinical (breast, bladder and prostate cancers) studies the use of the anticancer drug NSC-631570 was proven to be safe and highly effective, inhibiting protein synthesis in cancer cells, selectively accumulating in cancer tissue after a single intravenous administration and controlling cancer-induced metabolic imbalance. This drug inhibits metabolic processes in the tumor and causes metabolic disorders in cancer cells. Moreover, NSC-631570 induced the changes in certain amino acids concentrations in biological fluids and tumor tissue in animal models and cancer patients. These changes cannot be explained by metabolic amino acid disorders in cancer known so far. In this study the effects of NSC-631570 on blood plasma amino acids has been investigated. Blood was sampled from 10 healthy donors and 29 patients with different types of cancer (stomach, rectal, lung, breast, bladder, prostate, and leukemia). Comparison of NSC-531570 effects in plasma of healthy donors and cancer patients has shown, that this compoumd: 1) affects amino acids with positively charged (His, Arg) or not charged (Tyr, Thr, Gln) R-groups; 2) decreases concentration of His and increase the concentrations of beta-Ala and Tau. These changes depend on the concentration of NSC-631570 and the type of cancer. On the basis of the literature data and the results of our studies we suggest that Ukrain's biological actions in cancer are realized at least partly through selective interaction with amino acids, their derivatives, and plasma proteins. These data provide the background for the using Ukrain in the cancer detection and investigating the mechanisms of carcinogenesis.

Deoxyribonuclease Activity and Circulating DNA Concentration in Blood Plasma of Patients with Prostate Tumors

The DNase activity and circulating DNA (cirDNA) concentration in blood plasma of healthy donors, patients with chronic prostatitis, and patients with prostate tumors were analyzed. The concentration of the cirDNA from plasma was determined by PicoGreen fluorescent assay. DNase activity in blood was measured using the immunoassay based on the cleavage of a hapten-labeled 974-bp DNA substrate. The mean cirDNA concentration in the plasma of healthy donors was low (21 +/- 4 ng/mL total blood) and was accompanied by high DNase activity (0.17 +/- 0.04 U/mL blood). The mean cirDNA concentration was 90 ng/mL blood (10-234 ng/mL) in the patients with nonmalignant prostate tumors and 115 ng/mL blood (13-339 ng/mL) in those with prostate cancer. The mean DNase activity in blood plasma of the patients with prostate tumors was 0.06 U/mL blood (0-0.12 U/mL). The results obtained demonstrate that increased concentrations of cirDNA in blood of the patients with prostate tumors is accompanied by a decreased DNase activity, confirming our previous data that a low DNase activity in blood plasma of cancer patients is one reason for a high cirDNA concentration.

Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: preliminary study on a small group of recruits

Glycosaminoglycans in normal human plasma, mainly represented by chondroitin sulfates and heparan sulfates/heparin (HSGAGs), show a specific distribution in the Cohn-Oncley fractions of human plasma. In the present study we investigated their effects on coagulation. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated for extraction of glycosaminoglycans after extensive proteolysis; the anticoagulant activity in the extracted samples was measured by activated partial thromboplastin time (APTT). The effects of the samples containing HSGAGs on factor II and factor X activities, before and after treatment with heparinase I, were also measured. The molecular weight of HSGAGs was determined by polyacrylamide gel-electrophoresis. Cryoprecipitate and fraction I, fraction II+III, and fraction IV-1 (the fractions containing HSGAGs) prolonged the APTT, whereas fractions IV-4 and V had no effect on the APTT. Fractions containing HSGAGs showed effects on factor II and factor X activities that were sensitive to heparinase I treatment. The molecular weight of HSGAGs recovered in cryoprecipitate and fraction I was 15-18 kDa; that of HSGAGs recovered in fraction IV-1 was 12.0 kDa. In conclusion, these results demonstrate that HSGAGs of different molecular weight, endowed with anticoagulant activity, circulate in normal human plasma in association with specific proteins involved in the regulation of hemostasis; and that endogenous HSGAGs play a role in maintaining the antithrombotic/hemostatic balance in normal human plasma.

Immunochemical assay for deoxyribonuclease activity in body fluids

We have developed two microtiter plate assays to quantify the deoxyribonuclease activity in biological fluids. Both assays are based on hydrolysis of biotinylated and fluorescein-labeled DNA substrates, with subsequent immunochemical detection of non-digested DNA. The assay based on hydrolysis of 974 bp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers is more sensitive (0.05 U/ml) and convenient for quantifying the DNase activity in biological fluids than the assay based on hydrolysis of double-labeled 20 bp oligonucleotide. The DNase activity in urine and blood plasma of healthy donors was measured using the PCR product-based assay. Urine samples revealed greater activity, 1.49 ± 1.41 U/ml; blood plasma DNase I-like activity was 0.36 ± 0.20 U/ml. DNase II-like activity was not detected in the plasma samples. The data obtained confirm that DNase I-like enzymes are responsible for the majority of deoxyribonuclease activity in blood plasma.

Sensitive detection of plasma/serum DNA in patients with systemic lupus erythematosus

Objective: To optimize the use of the fluorochromic PicoGreen assay for the sensitive detection and quantification of double stranded (ds) DNA in plasma/serum samples of patients with systemic lupus erythematosus (SLE).Methods: Plasma/serum samples were obtained from SLE patients and normal healthy donors (NHD). Plasma/serum proteins were digested with proteinase K. DNA was subsequently purified using silica-based ion exchange micro columns and detected using the PicoGreen assay.Results: Sensitive detection of plasma/serum DNA is impaired by proteins: (1) Proteins caused background fluorescence in the PicoGreen assay. (2) Packaging of dsDNA in nucleosomes markedly reduced PicoGreen fluorescence. Therefore, we digested proteins using proteinase K and purified DNA before detection by PicoGreen assay. This procedure resulted in a detection limit for plasma/serum dsDNA of less than 1 ng/ml, and is therefore markedly more sensitive than previously described methods. We found that DNA concentrations are higher in serum than in plasma of healthy donors, suggesting artifactual DNA release during coagulation. In addition, we found higher levels of DNA in plasma and serum of a group of SLE patients compared to NHD.Conclusions: We have optimized the use of the PicoGreen assay for the ultrasensitive and reliable quantification of DNA in plasma/serum samples. This new method can be used in future studies to explore a possible correlation between circulating DNA levels and disease activity in patients with SLE.

Disseminated Mycobacterium avium Complex Infection in a Patient with Autoantibody to Interferon-.GAMMA.

A 54-year-old man was admitted to our hospital because of intermittent high fever, general malaise, cough and body weight loss after he had been examined and treated at the former hospital for 3 years without a definitive diagnosis and effective therapy. Bacterial examination on admission revealed Streptococcus pyogenes in peripheral blood, bone marrow aspirates and pleural effusion. Furthermore, Mycobacterium avium complex (MAC) was detected in sputum, gastric juice, bone marrow liquid, pleural effusion by acid-fast bacillus culture or polymerase chain reaction (PCR). Biopsy of the neck lymph node showed multiple granulomas and the presence of acid-fast bacilli. Administration of ABPC/SBT and later CLDM resulted in disappearance of Streptococcus pyrogenes after 2 weeks. We suspected of generalized immunodeficiency that might underlie the severe bacteremia/osteomyelitis and disseminated MAC infection. We tested interferon (IFN)-gamma production of peripheral blood mononuclear cells (PBMC) of the patient after phytohemagglutinin (PHA)-stimulation and found that these cells produced no or undetectable levels of IFN-gamma in the presence of autologous plasma while produced almost normal levels of IFN-gamma in the presence of healthy donor's plasma. Since this neutralizing activity was co-purified in the IgG fraction, the immunodeficient state of the patient seemed to be caused by autoantibody to IFN-gamma. As far as we know, this is the first report of a Japanese case of disseminated MAC infection presumably due to autoantibody to IFN-gamma.

Microparticles Are a Major Determinant of Thrombin Generation Measured by Technothrombin®TGA.

We have recently shown that a thrombin generation assay using low concentrations of phospholipids (3,2 mM; 20% PS) can discriminate patients with an increased risk for recurrent venous thromboembolism after cessation of anticoagulant therapy from those with a lower risk (Hron et al JAMA 2006). Patients with peak thrombin of ≥400nM had a ~3-fold higher relative risk of recurrence than those with peak thrombin <400nM. This difference was not explained by a larger proportion of patients with clotting factor abnormalities among those with high peak thrombin. We therefore investigated which other factors might influence peak thrombin generation. For this purpose 35 healthy male plasma donors were analyzed. Thrombin generation was measured in standard platelet poor plasma and in plasma made microparticle (MP) free by centrifugation for 120 minutes at 35,250 x g using low (3,2μM) and high (32μM ) phospholipids concentrations at the same tissue factor concentration (71,6 pM). For control purposes, peak thrombin was measured under similar conditions in 12 individuals each having either heterozygous FVLeiden mutation, being on vitamin K-antagonist therapy (VKA; mean INR=3.2) or having a lupus anticoagulant. In healthy plasma donors peak thrombin as generated by tissue factor and low phospholipids (RCL) in platelet poor plasma was 110±40 nM and increased to 306±99 nM in the presence of high phospholipids (RCH). When MP free plasma was used, however, peak thrombin dropped to 23±13 nM (RCL) and 109±43 nM thrombin (RCH), respectively. When MPs were re-added to MP-free plasma samples, a dose dependent increase in peak thrombin was found both for RCL and RCH reagents. At a MP concentration of 25,000 added per 1 ml MP free plasma, peak thrombin was 381±24 nM for RCL and 399±63 nM for RCH. The effects of MPs on peak thrombin using RCL were much more pronounced than the differences in peak thrombin seen between plasmas from individuals with FVLeiden, during anticoagulant therapy or with lupus anticoagulant as compared to controls (FVLeiden : 161±50 nM; VKA: 50±12 nM; lupus: 37±23 nM; controls: 110±40 nM). These results indicate that MPs contained in platelet poor plasma are a major determinant of peak thrombin in thrombin generation assays when low phospholipids are used and might cause increased peak thrombin levels found in patients with recurrent venous thromboembolism.

Circulating Proteasomes Are Functional and Have a Subtype Pattern Distinct from 20S Proteasomes in Major Blood Cells

20S proteasomes, the proteolytic core particles of the major intracellular protein degradative pathway, are potential disease markers because they are detectable in human plasma as circulating proteasomes and their concentrations are increased in patients suffering from various diseases. To investigate the origin of circulating proteasomes, we compared some of their features with those of proteasomes isolated from major blood cells. We isolated circulating proteasomes from the plasma of 2 patients with rheumatoid arthritis and 2 with systemic lupus erythematosus and from human plasma from healthy donors. We purified the proteasomes to apparent homogeneity and then used electron microscopy for imaging and chromatography for subtype spectrum analysis. We compared subtype results with those from 20S proteasomes purified from 4 major blood cell populations. We also tested proteasomes for enzymatic activity and immunosubunit content. Circulating proteasomes from plasma of healthy donors and from patients with autoimmune disease were found to have the same size and shape as erythrocyte proteasomes, be proteolytically active, and contain standard- and immunosubunits. Chromatography revealed 6 circulating proteasome subtype peaks in healthy donor plasma and 7 in patient donor plasma. Proteasomes from erythrocytes had 3 subtype peaks and those of monocytes, T-lymphocytes, and thrombocytes each had 5 different subtype peaks. Circulating proteasomes were intact and enzymatically active in plasma from healthy donors and from patients with autoimmune disease. Because the subtype patterns of circulating proteasomes clearly differ from those of proteasomes from blood cells, these cells cannot be regarded as a major source of circulating proteasomes.