High levels of cathepsins B, L and S in human seminal plasma and their association with prostasomes.

Semen is a heterogenous and complex fluid with different functions, some of them well known, others still obscure. The aim of this study was to investigate the presence of cathepsins B, S and L in human seminal plasma and their possible associations with other semen variables. Cathepsin B, L and S concentrations were measured in seminal plasma from 99 men utilising commercial ELISA kits. Seminal plasma cathepsin B was approximately 70 times higher, while the cathepsin L values were approximately 500 times higher and the cathepsin S values approximately 40 times higher in seminal plasma than in a group of serum samples. The study shows that seminal plasma contains high levels of cathepsins B, L and S. All three cathepsins were also bound to the surface of prostasomes.

Association of cystatin C with prostasomes in human seminal plasma.

It was recently elucidated that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, can also be secreted in association with exosomes. Accordingly, we wanted to investigate whether there is a secretory link between cystatin C and prostasomes in human seminal plasma. Cystatin C concentrations in seminal plasma from 50 men including 6 vasectomized men were measured by turbidimetry on an Architect Ci8200. Some of the seminal plasma samples were also analysed utilizing an Epics Profile XL-MCL cytometer. We found high concentrations of cystatin C in seminal plasma. The 2.5-97.5 percentiles, performed by bootstrap estimation, were 25.8 [95% confidence interval (CI): 22.3-29.4] to 77.0 mg/L (95% CI: 71.9-82.1). Cystatin C is present in approximately 50 times higher concentration in seminal plasma compared with blood plasma. There was no clear difference as regards seminal plasma content of cystatin C between vasectomized men and the rest of the group. Immunoblot analysis with chicken anti-cystatin C antibody revealed a firm association of cystatin C with prostasomes. Flow cytometric analysis demonstrated that cystatin C was linked to prostasomes also meaning an at least partial prostasomal membrane surface localization.

Rapid thawing of fresh-frozen plasma with radio wave-based thawing technology and effects on coagulation factors during prolonged storage at 4 degrees C.

BACKGROUND AND OBJECTIVES: With traditional techniques the thawing time for fresh-frozen plasma (FFP) is about 20-30 min. A new technology using radio waves, radio wave thawing device (RTD), was applied for thawing FFP. With this technology the thawing time can substantially be reduced. The new technique was compared to an established method using Heated Air Technology (HAT). Variables subjected to assessment were temperature after thawing and analysis of factor V (FV), factor VIII (FVIII), protein S. MATERIALS AND METHODS: Plasma was collected from 20 plasma donors. Each donation was aliquoted into two equal units of approximately 250 ml. The plasma units were frozen and stored below -75 degrees C. The thawing time was pre-set to two time periods, one for each group, 23 min for HAT and 7.5 min for RTD. Thawed plasma was stored at 4 +/- 2 degrees C as liquid plasma. Samples were collected before freezing, after thawing, 1 week and 2 weeks after thawing. RESULTS: The FV and FVIII levels were over 90% direct after thawing compared to before freezing values in both groups. At 2 weeks of storage the levels of FV and FVIII were approximately 70% and 50%, respectively, compared to before freezing values. Protein S levels decreased slightly during storage. No significant differences in the decline of quality were observed between the two groups. CONCLUSION: The new radio wave technology for thawing of FFP has a significant reduction of thawing time. The impact of thawing and storage on FV, FVIII, protein S does not significantly differ between HAT and RTD.

Coagulation parameters in apheresis and leukodepleted whole-blood plasma during storage.

BACKGROUND: Thawing fresh-frozen plasma (FFP) may cause delay in delivery, and one approach to circumvent this is to store plasma at +4 degrees C. Thawed plasma is commonly discarded after a few days of storage, owing to the assumption that coagulation factor activity decreases to clinically unacceptable levels. STUDY DESIGN AND METHODS: Eighteen apheresis plasma (AP) units were collected from blood donors. The collected plasma was divided into two equal parts: one part frozen at -74 degrees C as FFP and one part stored at +4 degrees C as fresh liquid plasma (FLP). Thirty-nine units of whole blood (WB) were collected from blood donors and leukodepleted by inline filtration, followed by plasma separation. Twenty plasma units were frozen at -74 degrees C as FFP and 19 plasma units were stored at +4 degrees C as FLP for 28 days. Plasma aliquots were collected before freezing and immediately after thawing FFP and before and during storage of FLP at Days 14 and 28. Factor (F)V, FVIII, D-dimers, and C1-esterase inhibitor levels were assessed. RESULTS: No significant differences in coagulation factor levels were assessed between FLP prepared from AP and FLP prepared from WB. FV and FVIII levels decreased on average 25 and 50 percent, respectively, at Day 14 of storage. C1-esterase inhibitor and D-dimers levels were not affected. CONCLUSION: Leukodepleted apheresis and WB plasma stored for 14 days retain sufficient levels of FV and FVIII activity for maintenance of normal hemostasis and could therefore be considered useful in selected clinical situations.

The role of coagulation and inflammation after angioplasty in patients with peripheral arterial disease.

PURPOSE: Restenosis remains a frequent complication after angioplasty in peripheral arterial disease. Inflammation plays a critical role in the vascular response to injury. Effective medical treatment to improve patency after angioplasty is still elusive. The aims of this prospective clinical study were to investigate changes in blood coagulation and inflammatory markers after angioplasty and their significance for restenosis. METHODS: Thirty-four patients with peripheral arterial disease underwent angioplasty of the iliac and superficial femoral arteries. Ten patients undergoing diagnostic angiography were included in the study as controls. Plasma levels of tissue factor, prothrombin fragment 1 + 2, D-dimer, P-selectin, C-reactive protein (CRP), and fibrinogen were analyzed before and after angioplasty. Patients were followed up with angiography after 6 months to assess restenosis. RESULTS: CRP was elevated the day after angioplasty (6.6 mg/l, p = 0.0001) and tended to peak after 1 week (11 mg/l, p = 0.09). There was a significant increase of D-dimer and P-selectin 1-4 hr after angioplasty (0.4 mg/l, p = 0.001 and 68 ng/ml, p = 0.05, respectively). None of the biochemical markers was a statistically significant predictor of restenosis. CONCLUSION: We have observed a much more prolonged inflammatory response than previously noted, but only minor changes in coagulation activity after angioplasty. The biochemical markers, before and after angioplasty, were not related to restenosis. Further studies are needed to delineate the molecular mechanisms behind these observations and their involvement in thrombosis and restenosis. If these pathways are further defined, improved treatment strategies, including antithrombotic treatments and statins, could be tailored to modulate postprocedural inflammation.

Haemostatic complications in haemodialysis patients: effect of type of vascular access and dialysis filter.

Impaired haemostasis is common in patients with end-stage renal disease, and may cause either thrombotic or bleeding complications. The purpose of this study was to assess whether plasma markers of coagulation activation in patients undergoing chronic haemodialysis (HD) can identify high-risk individuals, and to test the relevancy of type of vascular access or dialysis filter. We measured plasma levels of prothrombin fragment 1+2, fibrin D-dimers and tissue factor in 82 HD patients before and after dialysis. Clinical endpoints during the year following blood sampling were thrombosis in blood access, changes in blood access, other thromboembolic events, bleeding complications, ischaemic vascular disease, or death. We found elevated baseline levels of all three parameters in HD patients, compared to normal reference ranges. Plasma levels of all parameters (particularly fibrin D-dimers) were significantly higher in patients with prosthetic grafts and central venous dialysis catheters than in patients with native vessels. Patients with AV-fistulas or grafts who had bleeding complications (n=7) had significantly higher plasma levels of fibrin D-dimer and prothrombin fragment 1+2. Bleeding complications also occurred more frequently among the patients with prosthetic grafts (3/18) or central venous dialysis catheters (3/11) compared with those with grafts from native vessels (1/53). Other than a bleeding tendency, our data do no show any correlation between coagulation parameters and other clinical complications during haemodialysis. In conclusion, we found elevated plasma levels of markers of coagulation activation among HD patients. High levels of D-dimers and prothrombin fragment 1+2 correlated to bleeding diathesis instead of thromboembolism, and this tendency was most pronounced in patients with prosthetic grafts.

European Concerted Action on Anticoagulation (ECAA): multicentre international sensitivity index calibration of two types of point-of-care prothrombin time monitor systems.

A multicentre modified World Health Organization (WHO)-type international sensitivity index (ISI) calibration has been performed at 10 European Concerted Action on Anticoagulation (ECAA) national laboratories using non-citrated whole-blood on two point-of-care test (POCT) prothrombin time (PT) monitor systems, CoaguChek Mini and TAS PT-NC, using single lots of test cards/strips. The relevant species (human and rabbit) WHO international reference preparations (IRPs) were tested with the manual PT technique on citrated plasma from the same blood donations. The ISI was calculated from the slope of the orthogonal regression line relating log PT (POCT) to log PT (IRP). The mean ISI of the CoaguChek Mini system was 1.75 and 1.13 with the prothrombin time non-citrated Thrombolytic Assessment System (TAS PT-NC). With the CoaguChek Mini system, seven out of 10 calibrations exceeded the current 3% WHO recommended limit for the coefficient of variation (CV) of the slope with conventional PT testing, whereas with the TAS PT-NC system, it was eight out of 10. All the POCT calibrations had a CV of the slope <5%. It is suggested that this level of precision be adopted as the limit of acceptability of calibration of these monitor systems. In these circumstances, the modified WHO-type ISI calibration appeared to be satisfactory for the POCT whole-blood monitors.

Increases in circulating levels of monocyte-platelet and neutrophil-platelet complexes following hip arthroplasty.

Platelets and leucocytes are important effector cells of the haemostatic and inflammatory responses to tissue injury. To investigate the effects of surgical trauma on platelet activation (assessed by measuring levels of P-selectin and beta-thromboglobulin), leucocyte activation (CD11b expression) and leucocyte-platelet interactions (leucocyte-platelet complexes), 30 patients undergoing primary hip arthroplasty were studied before and at the end of surgery, and on days 1 and 10 post-operatively, using a whole-blood flow cytometry assay. The inflammatory response was followed by measurement of the levels of C-reactive protein and interleukin-6 in plasma, and the activation of coagulation was monitored by determination of prothrombin fragment 1+2 levels. On day 1 post-operatively a significantly increased expression of CD11b on monocytes was noted, but no direct correlation was found between monocyte activation and interleukin-6 production or C-reactive protein at this time point. The percentage of monocyte-platelet and neutrophil-platelet complexes was markedly increased on day 10 post-operatively compared with pre-operative levels, and levels of these complexes were significantly positively correlated with beta-thromboglobulin levels. Activation of coagulation (prothrombin fragment 1+2) on day 10 post-operatively was positively correlated with the extent of surgical trauma (duration of surgery, amount of blood loss) and with the increase in platelet activation (beta-thromboglobulin). In conclusion, hip arthroplasty induces platelet and coagulation activation, and also an inflammatory response that is maintained for more than 10 days post-operatively. This indicates an interaction between the immune and the haemostatic systems in the post-operative phase after hip arthroplasty.

[New in vitro analysis tested: bleeding time is still the best method for evaluation of primary hemostasis]. / Ny in vitro-analys prövad: blödningstid fortfarande bästa metod för test av den primära hemostasen.

The need is great for a simple, cheap and readily accessible method for the evaluation of primary hemostasis in work-ups at both out-patient clinics and units caring for surgical or intensive care patients. PFA-100 is a recently introduced instrument for in vitro testing of platelet function. We report experiences from Stockholm, Gothenburg and Malmo of PFA-100 measurements performed on samples from healthy controls and from patients with von Willebrand disease or platelet disorders. It is shown that the PFA-100 system has a high sensitivity for von Willebrands disease, while the sensitivity for hereditary platelet dysfunction is low. In its present design this new device could not replace the template bleeding time as a screening test for primary hemostasis.

Platelet storage in PAS-2 or autologous plasma: impact on functional parameters.

Currently, several platelet additive solutions for long-term platelet storage have been introduced. The aim of this study was to compare the deterioration of functional status of platelets stored for up to 5 days in autologous plasma (AP) only, with platelet stored in PAS-2, a salt solution containing acetate, citrate and sodium chloride. Change in platelet adhesion, aggregation and activation was measured by flow cytometric technique. In addition, beta-Thromboglobulin (beta-TG), lactate and glucose were determined. After 5 days of storage, the expression of P-Selectin was significantly higher, the production of lactate and the consumption of glucose were significantly lower, in platelets stored in PAS-2 than in autologous plasma. No significant differences were detected on day 5 between the two groups with regard to fibrinogen, von Willebrand factor binding capacity, or to beta-TG release. It can be concluded that neither storage medium was consistently better for the parameters tested. However, it must be emphasized that platelets stored in autologous plasma exhibited less lesion, in terms of P-Selectin expression compared with platelets stored in PAS-2.

Coagulation system activity before coronary artery bypass surgery for unstable angina.

OBJECTIVE: To measure coagulation activity immediately prior to coronary artery bypass surgery. Previous reports have shown that a hypercoagulable state and reduced fibrinolytic activity increase the risk of postoperative graft failure. DESIGN: Fifty patients with unstable angina and ongoing low-molecular-weight heparin antithrombotic treatment for a median of 4 days and 25 stable patients undergoing elective surgery were included. RESULTS: Antithrombin levels were significantly lower whereas fibrinogen and plasminogen activator inhibitor-1 levels were higher in the unstable patients than in the stable patients. Median preoperative levels of thrombin-antithrombin complex (TAT), prothrombin fragment1+2 (F1+2), fibrin D-dimers and beta-thromboglobulin did not differ significantly in unstable and stable patients. There were signs of activated coagulation with elevated levels of TAT and F1+2 before the operation in half of the unstable patients, who had had chest pain at rest within 48 h preceding the operation and also in one-third of patients undergoing elective surgery. CONCLUSION: A hypercoagulable state may be present in unstable as well as in stable angina pectoris patients accepted for coronary artery bypass surgery.

Thrombin inhibitors suppress the thrombin-thrombomodulin-mediated generation of activated protein C.

In the treatment of unstable coronary artery disease, direct thrombin inhibitors have shown no or only limited benefit as compared with heparin, despite theoretical advantages. One explanation may be that the direct thrombin inhibitors to a greater extent than heparin have an inhibiting effect on the generation and activity of activated protein C. In the present study, this hypothesis was tested in an in vitro, "purified" system, where human protein C underwent activation to activated protein C by the thrombin-thrombomodulin complex. Direct thrombin inhibitors, inogatran and hirudin, unfractionated heparin+antithrombin, or dalteparin+antithrombin, were added to the system before activation to evaluate their inhibitory effect on the generation of activated protein C. At inhibitor concentrations well below the achieved plasma levels in major clinical trials, the thrombin-thrombomodulin-mediated activation of protein C was inhibited by all the studied inhibitors in a dose-dependent manner, but, contrary to our hypothesis, to a greater extent by unfractionated heparin+antithrombin and dalteparin+antithrombin than by the direct thrombin inhibitors, hirudin and inogatran. Despite difficulties to draw conclusions for the in vivo situation, the in vitro inhibition, by all studied inhibitors, of the generation of activated protein C, found in this study may be a possible explanation for ongoing cardiovascular events despite adequate treatment with thrombin inhibitors, in patients with unstable coronary artery disease. This inhibition of the generation of activated protein C may also contribute to the rebound in cardiovascular events after withdrawal of effective antithrombotic treatment.

The risk of recurrent venous thromboembolism in carriers and non-carriers of the G1691A allele in the coagulation factor V gene and the G20210A allele in the prothrombin gene. DURAC Trial Study Group. Duration of Anticoagulation.

The results concerning the risk of recurrent venous thromboembolism (VTE) in carriers of the G1691A mutation in the coagulation factor V gene are not consistent and this risk in carriers of the G20210A polymorphism in the prothrombin gene has hitherto not been reported. We followed 534 patients for 48 months after their first episode of objectively documented VTE. The prevalence of the G1691A allele in 467 (87.5%) of the patients and in 207 controls was 25.3% and 8.2%, respectively, in heterozygote form and 2.4% and 0.5%, respectively, in homozygote form. The adjusted odds ratio (OR) for the first VTE was 4.4 (95% CI 2.6-7.8). The risk of recurrent VTE in heterozygotes was not statistically different from non-carriers (17.8% vs 17.6%), with 85% power to detect a hazard ratio of 2.35. Homozygotes had a significantly increased risk (p = 0.036) of recurrent VTE. The prevalence of the G20210A allele in 456 patients and 207 controls was 6.1% and 1.4%, respectively. The adjusted OR was 4.6 (95% CI 1.6-19.3) for the first VTE in 28 carriers of this polymorphism. The risk of recurrent VTE for these was not statistically different from non-carriers with an OR of 0.9 (95% CI 0.2-2.9).

The effect of a low molecular mass thrombin inhibitor, inogatran, and heparin on thrombin generation and fibrin turnover in patients with unstable coronary artery disease.

AIM: This study evaluated a novel specific thrombin inhibitor, inogatran, in comparison with unfractionated heparin, with regard to markers for coagulation activity in patients with unstable coronary artery disease. METHODS AND RESULTS: In the Thrombin Inhibition In Myocardial Ischaemia (TRIM) study patients were randomized to one of three different doses of inogatran or to unfractionated heparin, given intravenously over 72 h. In a subpopulation of 320 patients, markers for coagulation activity were measured at baseline, during and after the study infusion. Prothrombin fragment 1 + 2, indicating thrombin generation, decreased in the low, medium and high dose inogatran groups and in the heparin group during the first 6 h of treatment by 12%, 15%, 21% and 26%, respectively. From 6 h to 72 h after the start of infusion the levels changed by -7%, -6%, -4% and +34%, respectively. The increase in the heparin group continued after the infusion was stopped. Thrombin-antithrombin complex, also indicating thrombin generation, decreased by 0%, 2%, 18% and 22%, respectively, during the first 6 h of treatment. During the same period soluble fibrin, an intermediate in fibrin formation, increased both in the low and medium inogatran group by 9%, while a decrease by 4% and 18%, respectively, was seen in the high dose inogatran group and in the heparin group. Fibrin dissolution, as measured by fibrin D-dimer, decreased during the first 24 h of treatment by 20%, 18%, 18% and 20%, respectively. The first 24 h after discontinuation of infusion, fibrin D-dimer increased by 6%, 23%, 25% and 44%, respectively. After 72 h, at the end of infusion, patients treated with inogatran, to a larger extent than those given heparin, had suffered from death, myocardial infarction or refractory angina pectoris. After 7 days this trend was less marked. CONCLUSION: The more pronounced decrease in thrombin generation and fibrin turnover during the first 6 h of infusion, and the later increase in thrombin generation and fibrin turnover, in the heparin group, as compared to the inogatran groups, may be related to the lower clinical event rate during infusion with heparin compared with inogatran and the recurrence of ischaemic events, early after cessation of heparin infusion.

Genetic effects for plasma factor VII levels independent of and in common with triglycerides.

BACKGROUND: Coagulation factor VII has been demonstrated as a potential risk factor for cardiovascular disease. Both genes and non-genetic factors are related to plasma levels of factor VII. However, the extent to which genetic effects influence variability in plasma factor VII levels is unknown. Further, increased levels of plasma factor VII are associated with serum triglycerides, yet the reason for this association is not fully understood. METHODS AND RESULTS: Quantitative genetic analyses were applied to evaluate the relative importance of genetic and different environmental influences on plasma factor VII levels and to test the significance of genetic and environmental factors in common to factor VII and triglycerides in 215 pairs of middle-aged and elderly twins, of whom 104 were reared apart and 120 were women. Genetic influences were found to account for 57% of the individual differences in plasma factor VII levels, whereas shared-rearing and residual-familial environmental factors were not significant. Furthermore, a significant genetic correlation of 0.38 was found between factor VII and triglycerides, but the environmental correlation between these two measures was not significant. Genetic factors in common to factor VII and triglycerides explain about 7% of the total variance for factor VII. CONCLUSION: The present study suggests that there are substantial genetic influences on plasma factor VII levels. Furthermore, genetic effects explain the phenotypic association between factor VII and triglycerides.

Relationship between plasma fibrinogen and coronary heart disease in women.

Plasma fibrinogen is an independent risk factor for coronary heart disease (CHD) in men; however, its role in women is less clear. We examined the ability of plasma fibrinogen to predict CHD in a community-based, case-control study of women aged 65 years or younger living in the greater Stockholm area. Cases were all patients hospitalized for an acute coronary event between February 1991 and February 1994. Controls were randomly selected from the city census and were matched to cases by age and catchment area. Plasma fibrinogen was measured 3 to 6 months after hospitalization by using a fibrinogen assay based on fibrinogen polymerization time measurement. Of the 292 consecutive cases, 110 (37%) were hospitalized for an acute myocardial infarction and 182 (63%) for angina pectoris. The mean age+/-SD in both patients and controls was 56+/-7 years. Mean levels of plasma fibrinogen in patients and controls were 3.66+/-0.81 and 3.25+/-0.64 g/L (P<0.0001), respectively. The age-adjusted odds ratio (OR) for CHD in the highest versus the lowest quartile of plasma fibrinogen was 6.0 (95% confidence interval [CI], 3.5 to 10.4). After adjustment for age, cigarette smoking, body mass index, systolic blood pressure, total cholesterol, high density lipoprotein cholesterol, triglycerides, and educational level, the OR was 3.0 (95% CI, 1.6 to 5.5). Further adjustment for C-reactive protein yielded the same result. In both premenopausal and postmenopausal women, the multivariate-adjusted ORs were 7.0 (95% CI, 1.8 to 28.3) and 2.1 (95% CI, 1.0 to 4.4), respectively. These results provide evidence that plasma fibrinogen is associated with an excess risk of CHD in women.

Effects on the hemostatic system and liver function in relation to Implanon and Norplant. A prospective randomized clinical trial.

In this prospective randomized clinical trial, two long-term contraceptive implants were studied with respect to hemostasis and liver function in 86 healthy young women. The two implants used were Implanon, containing the progestagen etonogestrel (the biologically active metabolite of desogestrel) and Norplant, the implant containing the progestagen levonorgestrel. The results of the trial showed that both implants had similar small effects on the hemostatic system that are not suggestive of a tendency towards thrombosis. The effect on liver function was characterized by increases in total bilirubin and gamma-glutamyl transferase and decreases in alanine aminotransferase and aspartate aminotransferase.

Kinetics of bradykinin levels during and after leucocyte filtration of platelet concentrates.

OBJECTIVE: To measure bradykinin levels in platelet concentrates during and after leucocyte filtration. METHODS: Platelet concentrates were leukocyte depleted using three different leucocyte-depletion filters selected to represent filters with negative, positive or neutral charge, respectively. Bradykinin levels were analysed before, during and up to 90 min post-filtration. RESULTS: Significant levels of bradykinin were generated when negatively charged leucocyte depletion filters were used. However, we found a high variation between platelet concentrates prepared from different donors as well as within the same concentrate when this was divided into three aliquots. Generated bradykinin was rapidly degraded in the collection bag and no bradykinin was detectable 60 min post-filtration. CONCLUSION: Bradykinin generation is more pronounced when negatively charged leucocyte removal filters are used. Due to a rapid degradation of bradykinin in the storage bag, pre-storage filtration should minimise the risk of bradykinin related adverse reactions during transfusion with some filters.