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.

In vitro procoagulant and anticoagulant properties of Naja naja naja venom.

Bites by the Indian cobra (Naja naja naja) are common in India and Sri Lanka because of its close association with humans. Cobra venoms are complex and contain several toxic components, including neurotoxins that cause post-synaptic neuromuscular blockade with respiratory paralysis and even death. Bites may also cause extensive local necrosis by mechanisms not fully elucidated. Although no significant coagulopathy has been reported, N.n. naja venom can form blood clots in vitro by activating prothrombin as demonstrated by thrombin-specific chromogenic substrate. Scanning electron microscopy demonstrates that the clots formed by venom lack the thin fibrin strands of normal blood clots formed by thromboplastin or glass contact. Rheometry shows that clots formed by venom have abnormally low elasticity over an extended period and then, as the platelets contract, a retarded and more feeble increase in elasticity. Purified N.n. naja venom PLA2 inhibits platelet aggregation in PRP and explains the decreased clot retraction and retarded and compromised elasticity build up. The present study shows that the PLA2 and the prothrombin activator from N.n. naja venom have effects on haemostasis and blood clotting, although such effects are not observed systemically in envenomed humans.

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.

Isocitrate as calcium ion activity buffer in coagulation assays.

BACKGROUND: Ca(2+) activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca(2+) activity at approximately 1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca(2+) buffering improves diagnostic efficacy in global diagnostic coagulation tests. METHODS: Buffering was investigated by mixing CaCl(2) and 11 candidate compounds and determining Ca(2+) activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca(2+) activity to approximately 1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved. RESULTS: Two suitable Ca(2+) buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca(2+) buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca(2+) activity of 1.60 +/- 0.07 mmol/L, compared with 2.73 +/- 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 +/- 5.7, compared with 3.6 +/- 5.0 (P <0.005) for the original APTT. CONCLUSIONS: Isocitrate can be used to buffer Ca(2+) activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca(2+) activity shows signs of improved diagnostic efficacy.

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.

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.

Clinical evaluation of a diagnostic strategy for deep venous thrombosis with exclusion by low plasma levels of fibrin degradation product D-dimer.

Clinical research studies have indicated the possibility of diagnostic strategies for deep venous thrombosis (DVT), strategies which include a step where the diagnosis is excluded by low or undetectable plasma levels of fibrin degradation product D-dimer. In collaboration with two local hospitals in Sweden, three implementations of such a strategy are evaluated in this study. Procedures 1, 2 and 3 differed in the method for D-dimer determination, i.e. latex agglutination, immunofiltration and both, respectively. The evaluated procedures were performed in parallel and compared with the current procedure in the different hospitals. At both hospitals, the current procedure stipulated mandatory phlebography and laboratory analysis of acute coagulation status and routine haematology with report-back time of 2 h. Within the 2 h the hospitals' clinical chemistry laboratories also determined plasma D-dimer by the two methods. Of 180 patients enrolled in the study, phlebography was successful in 155 and unsuccessful in 25. The phlebographies revealed 47 proximal DVT, 13 distal DVT and 95 no DVT. With Procedure 1, 53 patients (29%) were excluded in the D-dimer step. For these patients, 47 successful phlebographies revealed one proximal DVT and two distal DVT. With Procedure 2, 71 patients (39%) were excluded. For these patients, 65 successful phlebographies revealed two proximal DVT and four distal DVT. With Procedure 3, 44 patients (24%) were excluded. For these patients, 41 successful phlebographies revealed two distal DVT. The negative predictive values of the D-dimer exclusion step, with 95% confidence intervals given within parentheses, were 96% (88-100%), 91% (84-98%) and 95% (89-100%) for Procedures 1, 2 and 3, respectively. The evaluation demonstrated that the diagnostic potential of D-dimer revealed in research studies can be achieved in clinical practice. The study also indicated that the positive diagnostic value of high levels of D-dimer may be of use in finalizing the diagnosis in the 14% of patients for whom phlebography is unsuccessful.

Mobilization of fibrinolytic enzymes in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy and their relation to radiological destruction.

OBJECTIVE: To study the role of fibrinolytic enzymes in inflammatory joint diseases with different types of joint destruction. METHODS: Concentrations of the plasminogen activators pro-urokinase, tissue plasminogen activator, plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), plasminogen, and fibrin split product D-dimer in synovial fluid (SF) and blood plasma of 29 patients with rheumatoid arthritis (RA) were compared with the concentrations of 18 patients with spondyloarthropathy (SpA). Levels of the fibrinolytic components were also related to radiological destruction assessed using the Larsen grading system. RESULTS: Patients with RA had significantly higher PAI-1 antigen levels and PAI-1 activity in SF than patients with SpA. Plasma levels of PAI-1 antigen and D-dimer were significantly higher in RA than in SpA. There was also a tendency of lower tPA activities in SF and plasma of patients with RA. Joint destruction correlated significantly with increasing PAI-1 antigen and with decreasing plasminogen in SF when results from all patients were pooled. A significant negative correlation between plasma PAI-2 antigen and Larsen grade was also found. CONCLUSION: Our results indicate a possible association between joint destruction and mobilization of fibrinolytic enzymes in SF.

Tissue plasminogen activator, plasminogen activator inhibitor-1 and von Willebrand factor in rheumatoid arthritis.

Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), all of endothelial origin and active in the haemostasis, were analysed in 74 patients with rheumatoid arthritis. The concentrations were related to extra-articular disease and to the incidence of thromboembolic events (TE) registered in a 2-year follow-up period. Patients with extra-articular disease had a significant increase in PAI-1 activity and reduced tPA release in the venous occlusion test. von Willebrand factor, PAI-1 and also haptoglobin and triglycerides were significantly increased in the group of patients who later suffered from TE. In a multiple regression model, in which cholesterol, triglycerides and lipoprotein (a) showed significant association with TE, vWF had the strongest additive explanatory value. No distinct acute phase pattern of PAI-1 was found in any patient subgroup.

Oat husk fiber decreases plasminogen activator inhibitor type 1 activity.

Eleven healthy subjects volunteered to participate in a fiber supplement study and were instructed to consume oat husk tablets in addition to their regular diets. During a 2-week experimental period the subjects consumed 10 g oat husk per day. Blood samples were collected before breakfast between 8:00 and 9:00. As compared to baseline, 10 g oat husk supplementation per day resulted in a reduction of plasma plasminogen activator inhibitor type 1 (PAI-1) activity (p < 0.05). Except for an increase in glucose, no other statistically significant deviation from baseline was observed in measured blood parameters; tissue plasminogen activator activity, prourinary plasminogen activator, coagulation factor VII, total cholesterol, HDL cholesterol, LDL cholesterol, lipoprotein (a), triglycerides and insulin. A 6-week washout period returned the PAI-1 activity level to baseline.

Diet-induced changes in glucose and triglycerides are associated with changes in plasminogen activator inhibitor levels.

Twenty-four healthy female subjects volunteered to participate in an experiment in which they maintained a high-fat/low-carbohydrate (CHO) diet followed by a low-fat/high-CHO diet or vice versa in a cross-over study design. In bivariate correlation analysis, only glucose (r = 0.52, p less than 0.05) and triglycerides (r = 0.53, p less than 0.05) correlated with changes in plasminogen activator inhibitor type 1 (PAI-1) activity. A second-degree polynomial response surface model suggested that transition from a high-fat/low-CHO diet to a low-fat/high-CHO diet is associated with reduced levels of PAI-1 provided that glucose and triglyceride levels are not elevated by more than 1.2 and 0.5 mmol/l, respectively.

Effects of methylamine and heparin on a rapid chromogenic assay of C1-esterase inhibitor in plasma.

We describe a rapid assay of C1-esterase inhibitor (C1-inh) activity in plasma. After adding purified C1s serine protease (EC 3.4.21.42) in excess to plasma, we determine the residual C1s activity towards a new chromogenic tripeptide, CH3CO-Lys(CbO)-Gly-Arg-pNA. Optimal conditions include the addition of methylamine (final concentration 0.12 mol/L) to reduce the potential inhibitory capacity of alpha 2-macroglobulin towards C1s and the addition of heparin (final concentration 3000 int. units/L) to enhance the reaction of C1s with C1-inh. The correlation with C1-inh antigen concentrations in plasma was excellent. The estimated interassay CV was 4.3%, whereas the intra-assay CV was 2.0% for activity concentrations within the range of normal individuals (means +/- 2 SD: 70-124%), 1.3% at lower concentrations. The method is more convenient, rapid, and precise than previous methods, and C1-inh activity in plasma can be assessed within 30 min. We found that concentrations of C1-inh in plasma were low during open-heart bypass surgery.

Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal tPA activity.

Tissue plasminogen activator (tPA) activity in blood and blood plasma has been difficult to determine because it is unstable, especially in subjects with high levels of plasminogen activator inhibitor type 1 (PAI-1). We have attempted to stabilize the tPA activity by collecting 9 volumes of blood in 1 volume acidic citrate buffer which immediately lowers the pH of the blood. At final blood pH of 4.9 to 6.3, consistent and high tPA activity levels were found. One acidic citrate buffer composition, 0.5 mol/l citrate buffer pH 4.0, resulted in final blood pH 5.5, tPA half-life of 10 days and an acceptably low degree of haemolysis. This anticoagulant composition was selected for more extensive evaluation and was used to collect blood plasma samples from 29 volunteers in the morning after a 10-minute bed rest. Basal tPA activity, mean and SD, was 0.47 +/- 0.21 IU/ml. After donating of the first blood sample, 10 of the volunteers entered into a 24-hour fast after which they donated a second sample. During the fast, the basal tPA activity increased from 0.34 +/- 0.21 to 0.47 +/- 0.23 IU/ml (p = 0.05) and the PAI-1 activity decreased from 11.4 +/- 11.6 to 6.3 +/- 8.9 U/ml (p = 0.04).

Immunoreactivity of tissue plasminogen activator and of its inhibitor complexes. Biochemical and multicenter validation of a two site immunosorbent assay.

An enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 81% for free tPA and tPA in complex with PAI-1, PAI-2, alpha 2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about 100%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasma, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Fibrinolytic variables are related to age, sex, blood pressure, and body build measurements: a cross-sectional study in Norsjö, Sweden.

Levels of the fibrinolytic variables, tissue plasminogen activator (tPA) antigen concentration and plasminogen activator inhibitor (PAI-1) activity, were measured in a cross sectional sample of 260 subjects aged 30, 40, 50, or 60 years. There was a significant increase of tPA with age in both sexes, but PAI-1 increased only in women. Linear regression analysis was used to assess relations between tPA or PAI-1 and the anthropometric data. In men, tPA levels were related to body mass index and waist-to-hip ratio, whereas in women, it was also related to systolic and diastolic blood pressures and with abdominal or triceps skinfold thicknesses. PAI-1 levels were related to body mass index and waist-to-hip ratio in men, and in women it was in addition related to systolic and diastolic blood pressures and to abdominal and triceps skinfold thicknesses. These data offer new insight into pathophysiological mechanisms whereby age, sex, blood pressure, and body composition variables such as body mass index or waist-to-hip ratio, might act as cardiovascular risk factors.

An affinity gel for the inhibition, binding, and isolation of serine proteases.

A preparation procedure for gels for the specific binding and inhibition of serine proteases is described. Phosphoryl trifluoride was synthesized and reacted with two different types of agarose gels, a crosslinked agarose (Sepharose CL-4B) and an agarose containing spacer arms with terminal vicinal-diol groups (a hydrolyzed epoxy-activated Sepharose 6B). The phosphoryl difluoride groups coupled to the gels were, in both cases, further modified by treatment with isopropanol to obtain isopropyl fluorophosphate groups covalently bound to the matrix. It was found that both modified gels absorbed and inhibited plasmin, but that the modified gel with spacer arms was markedly more efficient.

Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single-point poly-D-lysine stimulated assay.

A two stage method for determination of plasminogen activator inhibitor (PAI) activity in blood plasma is described. In the first stage, an excess of single-chain tissue plasminogen activator (t-PA) is added to plasma. In the second stage, the residual t-PA activity is determined with a plasminogen/chromogenic plasmin substrate assay utilizing poly-D-lysine as a t-PA stimulator. The method proved accurate since it correctly determined the PAI activity (range 0 to 110U/mL) in citrated plasma samples with levels established by time course analysis of t-PA inhibition and by titration with t-PA. Furthermore, correlation was excellent (r = 0.97) with the method of Chmielewska et al Thromb. Res. 31, 427-436, 1983. Plasma samples with increased platelet factor 4, indicative of platelet release, did not show increased levels of PAI activity.