Coagulation activation by lipopolysaccharides.

Lipopolysaccharides at approximate plasma reactivities >3 ng/mL or beta-glucans at >0.5-1 Amicrog/mL are toxic for human blood; lipopolysaccharide interacts with membrane components of susceptible cells (eg, monocytes) activating phospholipase A(2) that destroys the cell membrane. Cell fragments (microparticles or DNA) possess polynegative niches that activate intrinsic hemostasis. Pathologic disseminated intravascular coagulation arises. Blood vessels are obstructed by disseminated thrombi, and vital organ areas become ischemic. Multiorgan failure threatens life of the patient. Diagnosis and therapy of pathologic disseminated intravascular coagulation is of extreme clinical importance. For early diagnosis of pathologic disseminated intravascular coagulation, specific activation markers of coagulation (eg, plasmatic amidolytic thrombin activity) or the plasmatic lipopolysaccharide or glucan reactivity can be measured. A new treatment target might be kallikrein or factor XIIa; 10 to 20 mM arginine is the approximate 50% inhibitory concentration against the contact phase of coagulation. The complex interaction between cell fragments and hemostasis causes pathologic disseminated intravascular coagulation in sepsis.

Intrinsic hemostasis activation by freezing and thawing of plasma.

Low-grade contact activation of hemostasis is clinically relevant. Freezing/thawing of plasma was studied in the intrinsic coagulation activity assay. Normal plasmas were frozen at -80 degrees C or -20 degrees C and thawed at 37 degrees C or 23 degrees C. These plasmas and unfrozen samples were activated by SiO2 -CaCl2. Freezing/thawing of normal plasma induced about 100-fold more thrombin activity at 5 minutes coagulation reaction time than the respective unfrozen samples. Freezing at -80 degrees C induces more artificial changes than freezing at -20 degrees C. In 9 of 10 plasmas of patients receiving coumarin, nearly no additional thrombin is generated within a 12-minute coagulation reaction time. Minor procoagulant changes of plasma might be dangerous in patients with insufficient liver function, who might not tolerate a therapy with fresh frozen plasma, which behaves as a procoagulant because of its matrix changes. The intrinsic coagulation activity assay allows the measurement of low-grade contact activation of frozen/thawed plasma.

Monitoring of functional plasminogen in the blood of patients on fibrinolytics.

There are no reliable data on functional plasminogen in the blood of patients receiving fibrinolytic treatment. Here, artifactual in vitro changes of functional plasminogen were prevented by arginine stabilization blood samples of myocardial infarction patients: 12 received 36.4 mg reteplase in bolus, and 1 patient received 100 mg tissue plasminogen activator in continuous infusion. Arginine (1.5 M, 1.3 mL, pH 8.7) was used to stabilize 2.6 mL ethylenediaminetetraacetic acid-blood. The arginine-stabilized plasma was analyzed with a functional oxidative assay for plasminogen. Functional plasminogen decreased within 2 minutes of reteplase treatment by about 40% and by about 80% after 60 minutes. Lowest plasminogen concentrations were found in plasmas with highest plasmin activities. Chloramine oxidation of purified Glu-plasminogen increased its activation by urokinase up to 3-fold. Arginine stabilization allows reliable determinations of functional plasminogen in the blood of patients receiving fibrinolytics, enabling the rapid diagnosis of prothrombotic plasminogen consumption. The present findings support the profibrinolytic action of chloramines.

The recalcified coagulation activity.

Thrombin activity generated after plasma recalcification is of analytical and clinical interest. Fifty microliters of citrated plasma was recalcified with 5 microL of 250 mM CaCl( 2). After 0 to 90 minutes (37 degrees C) 50 microl 2.5 M arginine, pH 8.6, was added. After 20 minutes, thrombin was chromogenically quantified. In normal recalcified plasma, the generated thrombin activity is about 0.1-0.2 IU/ml (37 degrees C) when fibrin generation starts. Pooling of normal plasmas increases the generated thrombin activity about 3-fold. Plasmas of patients on heparin or coumarin generate about 10-fold less thrombin activity. Freezing of pooled plasma at -20 degrees C and thawing at room temperature or 37 degrees C increases thrombin generation approximately 1.5- or 2-fold, respectively. Only thrombin activities in the ascending part of the thrombin generation curve (RECA-t(2)/RECAt( 1)>1) are valid. So a prothrombotic state in blood or plasma can be diagnosed.

Kallikrein activates prothrombin.

Kallikrein is a multitalented enzyme in hemostasis and inflammation. Normally, kallikrein is formed in intrinsic hemostasis and activates factor XII. A total of 10 microL of 0 to 100 microg/mL human plasma kallikrein in 6% human albumin-PBS were incubated with 90 microL 111.1 microg/mL prothrombin in 6% human albumin in absence and presence of 23 mM Ca(++). After 0 to 64 minutes (37 degrees C), 100 microL of 2.5 M arginine, pH 9, were added. Fifty microliters of 0.72 mM HD-CHG-Ala-Arg-pNA in 1.36 M arginine were added and increase in absorbance at 405 nm was determined. Within 8 minutes (37 degrees C), 1 microg/mL kallikrein, ie, 2.5% of the normal plasmatic prekallikrein concentration, generates approximately 3 mIU/mL thrombin in absence and 27 mIU/mL thrombin in presence of Ca(++). Kallikrein can directly activate prothrombin; there is a shortcut in the intrinsic hemostasis system that generates catalytic amounts of thrombin without following the known intrinsic clotting pathway.

In vitro simulation of thrombolysis inhibition.

Hyperfibrinolysis is a serious clinical complication. The inhibitory concentrations 50% of antifibrinolytics were analyzed in the microtiter plate clot lysis assay, using 50 microL of plasma clots, 10 microL of antifibrinolytic drug, 10 microL of 354 IU/mL (final) urokinase, 4.46 microg/mL (final) tissue-type plasminogen activator or 0.6 mg/mL plasmin, and 50 microL of pooled normal plasma as clot supernatant. The inhibitory concentrations 50% of lysine against urokinase or tissue-type plasminogen activator is 2.0 or 4.2 mM, against epsilon-amino-caproic acid 0.7 or 1.5 mM, against tranexamic acid 0.03 or 0.17 mM, respectively. The inhibitory concentrations 50% of lysine, epsilon-amino-caproic acid, or tranexamic acid against plasmin is 7.4, 0.4, or 0.04 mM. The inhibitory concentrations 50% of aprotinin against urokinase or tissue-type plasminogen activator is about 60 KIU/mL, against plasmin 19 KIU/mL. Lysine might be a new antifibrinolytic drug with a clinically interesting rapid pharmacokinetic. This data help correct dosing of antifibrinolytics to patients with hyperfibrinolysis.

In vitro simulation of extremely activated thrombolysis.

A life-threatening thrombus in massive pulmonary embolism has to be eliminated within minutes. Extremely activated plasmatic fibrinolysis destroys such thrombi in time: 50 microL plasma clots were incubated with urokinase or tissue-type plasminogen activator and 50 microL pooled normal plasma. The microtiter plate clot lysis assay was performed. The time point at which 50% of the clot has been lysed is 4 minutes for 8333 IU/mL urokinase or an equimolar concentration of tissue-type plasminogen activator (52498 IU/mL = 105 microg/mL). The effective dose 50% at 5 minutes lysis time is about 800 nM (4320 IU/mL) urokinase or (27220 IU/mL = 54 microg/mL) tissue-type plasminogen activator. Addition of plasminogen to the plasmatic clot supernatant improves thrombolysis if 65 IU/mL of urokinase acts for 10 minutes. The risk for severe intracranial hemorrhage in massive thrombolysis might be much lower than the lethality of a massive pulmonary embolism. Extremely activated plasmatic thrombolysis could be clinically indicated.

The fibrinogen functional turbidimetric assay.

Hitherto, clinical fibrinogen methods were based on coagulation seconds, with assay conditions not similar to a plasma milieu. The fibrinogen functional turbidimetric assay included 50 microL citrated plasma + 100 microL 300 mIU/mL thrombin, 400 microg/mL polybrene, and 6% albumin-phosphate-buffered saline; an increase in absorbance at 405 nm/5 min at room temperature (or 2 minutes at 37 degrees C) was observed. In all, 6% albumin in the fibrinogen functional turbidimetric assay reagent abolishes falsely elevated fibrinogen to fibrin turbidity in hypoproteinemic plasma samples. This assay can detect fibrinogen activity of 250% to 300% of normal, the lower detection limit being 7% of normal (0.2 g/L). The normal range of this assay is 100% +/- 20% (mean value +/- 1 SD; coefficient of variations <4%). This assay imitates fibrinogen to fibrin conversion in clotting blood plasma; it is independent of plasmatic albumin or heparin and can be performed everywhere. This assay has a diagnostic value in pathology-disseminated intravascular coagulation and in assessing risk for atherothrombosis.

The extrinsic coagulation activity assay.

A chromogenic assay for the tissue factor-mediated thrombin generation was developed, the extrinsic coagulation activity assay: 50 microL citrated plasma is incubated with 5 microL tissue factor in 6% albumin and 250 mM CaCl2. After 1-minute (37 degrees C) coagulation reaction time, (extrinsic coagulation activity assay with 1-minute coagulation reaction time; generating normally about 1 IU/mL thrombin) 100 microL 2.5 M arginine is added to stop hemostasis activation. Generated thrombin is then chromogenically quantified. The normal extrinsic coagulation activity assay range is 100% +/- 20%. Extrinsic coagulation activity assay in plasma of patients on heparin or coumarines is about 10-fold lower. Advantages of extrinsic coagulation activity assay: normal range of extrinsic hemostasis is truly represented, patients prone to hyper-activated extrinsic pathway are detected, anticoagulants result in respective test inhibition, fibrinogen/fibrin concentration does not artefactually alters the test result, plasma matrix is not changed significantly in the assay, and assay results are IU/mL thrombin or % of normal, which can be measured by every normal photometer.

Thrombin generation by hemolysis.

Hemolysis is the fragmentation of erythrocytes into microparticles (Hb-MP). Clinical hemolysis can result in a severe procoagulant state. The influence of Hb-MP on thrombin generation was quantified. Unfrozen citrated normal plasma (five donors) was supplemented with 0 or 1 g/l Hb-MP obtained through erythrocyte destruction by hypotonic lysis, freezing/thawing, or blood oxidation with 1 or 2 mmol/l chloramine-T. Pooled normal plasma was supplemented with 0-10 g/l Hb-MP and with 0-1 IU/ml low-molecular-weight heparin (dalteparin). Samples (50 microl) were tested in the recalcified coagulation activity assay. At 10 min coagulation reaction time the hypotonic lysis of erythrocytes appears to be the most procoagulant condition, followed by twice freezing/thawing, three times freezing/thawing, and once freezing/thawing. Oxidation of whole blood with 1 or 2 mmol/l chloramine-T decreases thrombin generation by about 20 or 50%, respectively. The thrombin generation in 1 mmol/l chloramine-T or 2 mmol/l oxidized plasma decreases by about 70 or 85%, respectively. The 50% inhibitory concentrations of low-molecular-weight heparin against recalcified thrombin generation are 0.01, 0.025, or 0.035 IU/ml for plasma supplemented with 0, 0.1, or 1 g/l Hb-MP, respectively. The recalcified coagulation activity assay allows one to quantify thrombin generation in critical hemolytic samples. It is suggested to find the appropriate pharmacologic dose of low-molecular-weight heparin.

Inhibition of thrombin generation in recalcified plasma.

Eight inhibitors of thrombin generation were compared in recalcified unfrozen plasma. Individual or pooled normal citrated plasma was supplemented on polystyrol flat-bottom wells (23 degrees C) with increasing concentrations of low-molecular-weight heparin, heparin, danaparoid, fondaparinux, hirudin, argatroban, corn trypsin inhibitor, or aprotinin. Thrombin was generated by addition of 5 microl fresh 250 mmol/l CaCl2 to 50 microl plasma in polystyrol flat-bottom wells and incubation for 20 min at 37 degrees C (recalcified coagulation activity assay). Arginine stopped hemostasis activation and then the generated thrombin activity was specifically quantified. The approximate 50% inhibitory concentrations of plasmatic anticoagulants for individual or pooled normal plasma are, respectively, 0.6 or 3.7 mIU/ml low-molecular-weight heparin, 0.3 or 1.6 mIU/ml heparin, 0.7 or 6.1 mU/ml danaparoid, 0.023 or 0.18 microg/ml fondaparinux, 75 or 230 pg/ml hirudin, 0.026 or 0.24 microg/ml argatroban, 1 or 2 U/ml corn trypsin inhibitor, and 2 or 4 KIU/ml aprotinin. The 50% inhibitory concentration values for corn trypsin inhibitor or aprotinin at plasmatic concentrations above 4-100 U/ml might increase pathologically the thrombin generation. The recalcified coagulation activity assay is a sensitive method to measure prothrombotic tendencies of blood or subtle concentrations of any plasmatic anticoagulant. It is suggested to analyze the individual patient's sensibility to certain plasmatic anticoagulants.

The anticoagulant capacity of plasmatic unfractionated heparin decreases at 23 degrees C.

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are important clinical anticoagulants. As polynegative molecules they are potential triggers of the contact phase of coagulation. An incubation temperature lower than the physiological 37 degrees C favours intrinsic haemostasis activation by the polynegative molecule SiO2. The efficiency of UFH and LMWH after a plasmatic preincubation at 37 or at 23 degrees C is therefore studied. Samples (150 mul) of unfrozen pooled normal plasma supplemented with 0, 0.01, 0.1, or 1 IU/ml heparin or dalteparin in 5-ml polystyrole tubes were incubated for 10-70 min at 37 or at 23 degrees C. The extrinsic coagulation activity assay (EXCA) was then performed. Preincubation at 37 degrees C of 0.1 IU/ml plasmatic UFH does not result in any thrombin generation in EXCA-1, whereas preincubation at 23 degrees C results in a thrombin generation of about 0.1 IU/ml thrombin. Plasmatic UFH (0.01 IU/ml) at 23 degrees C acts nearly half as efficiently as 0.01 IU/ml plasmatic LMWH. Polynegatively charged niches particularly in the larger UFH molecule might trigger the contact system of haemostasis, especially at 23 degrees C. In contrast, the anticoagulant capacity of LMWH does not change significantly with temperature.

The efficiency of anti-activated factor X and anti-activated factor II anticoagulants.

Six thrombin-generation inhibitors or thrombin inhibitors were compared in the extrinsic coagulation activity assay (EXCA), where the normal thrombin generation is about 1 IU/ml within 1 min (37 degrees C). Unfrozen pooled normal citrated plasma was supplemented on flat-bottom wells (23 degrees C) with increasing concentrations of dalteparin, danaparoid, heparin, fondaparinux, hirudin, or argatroban. To 50 microl plasma, 5 microl of 1.5 ng/ml tissue factor, 6% bovine serum albumin, and 250 mmol/l CaCl2 were added. After 1 and 2 min coagulation reaction time at 37 degrees C (EXCA-1 and EXCA-2), 100 microl of 2.5 mol/l arginine and 0.16% Triton X 100, pH 8.6, were added. After 3 min (23 degrees C), 25 microl of 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, were added, and the linear increase in absorbance with time was determined at 405 nm. The 50% inhibitory concentrations of plasmatic anticoagulants tested in the EXCA-1 (37 degrees C) were 0.025 IU/ml dalteparin, 0.13 U/ml danaparoid, 0.12 IU/ml heparin, 1.3 microg/ml fondaparinux, 2.4 ng/ml hirudin, and 1 microg/ml argatroban. From the 50% inhibitory concentration of hirudin it can be concluded that inhibition of about 30 mIU/ml thrombin halves the normal EXCA-1 value (i.e. if about 0.1 IU/ml thrombin are inactivated, then thrombin cannot self-amplify its generation 10-fold). The efficiency of any clinically used plasmatic anticoagulant can be monitored in the EXCA.

Determination of the global fibrinolytic state.

Fibrinolysis consists of a plasmatic part and a cellular part. A rapid global assay for plasmatic fibrinolysis is the fibrinolysis parameters assay (FIPA). Cellular fibrinolysis is measured by testing the clot lysis capacity using the microtitre plate clot lysis assay with polymorphonuclear neutrophils (CLA-PMN). Individual citrated plasma or pooled normal plasma (50 microl) of 232 patients was recalcified, incubated for 90 min at 37 degrees C, oxidized with 0 or 1.5 mmol/l (final concentration) chloramine-T, and supplemented with 50 microl respective polymorphonuclear neutrophil plasma. The turbidity of the clots was measured at 405 nm after 12 h and 60 h (37 degrees C). Plasma (50 microl) was also incubated with 5 microl of 100 IU/ml urokinase, 6 mmol/l tranexamic acid, 6% human albumin for 10 min (37 degrees C). Then 100 microl of 0.5 mmol/l Val-Leu-Lys-pNA in 2.45 mol/l arginine, pH 8.6, was added and the increase in absorbance with time was measured. The different CLA-PMN assay versions correlated with each other with r = 0.543-0.782. Cellular fibrinolysis (34 +/- 30% lysis; normal: 25 +/- 10%) did not correlate with the FIPA (72 +/- 27%; normal: 100 +/- 15%), prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, or the blood counts of thrombocytes, leukocytes, or polymorphonuclear neutrophils. Chloramine (1.5 mmol/l) oxidation of the microclots favours their fibrinolytic breakdown, especially if lysis-resistant microclots are oxidized. The FIPA and CLA-PMN are new economical tests for the fibrinolytic state in patient blood.

Influence of coagulation factors on intrinsic thrombin generation.

The intrinsic coagulation activity assay (INCA) is a new thrombin-generation test that imitates the intrinsic pathway of blood coagulation. The aim of the present study was to investigate the influence of the main coagulation factors on the INCA. The INCA was performed with citrated plasma samples supplemented with different amounts of fibrinogen. The INCA and activated partial thromboplastin time determination were performed with factor-depleted plasmas and with mixtures of depleted plasmas with normal plasma. Supplemented purified fibrinogen resulted in a decrease of intrinsic thrombin generation (50% inhibitory concentration = 0.8 g/l). The INCA depends on the intrinsic factors (factors VIII, IX, XI and XII) and on the factors of the common pathway (factors II, V and X): for normal thrombin generation, at least about 50% of normal factor II is necessary. For the majority of factors, the sensitivity of the INCA appears to be approximately one order of magnitude better than that of the activated partial thromboplastin time. The INCA allows one to diagnose defects in the intrinsic coagulation system and might be a useful test to support development and characterization of new drugs targeted at the intrinsic generation of thrombin.

Influence of coagulation factors on extrinsic thrombin generation.

The extrinsic coagulation activity assay (EXCA) is a new thrombin generation test for the tissue factor pathway of coagulation. The EXCA was performed with 10 parts citrated plasma of different contents of fibrinogen. One part tissue factor, 250 mmol/l CaCl(2), generating about 1 IU/ml thrombin within 1 min (37 degrees C). After 0-30 min 2.5 mol/l arginine (pH 8.6) Generated thrombin was detected by addition of CHG-Ala-Arg-pNA and measurement of triangle upA/t. The EXCA is dependent on factors 10% of the factor VII norm in the sample achieves 70-80% of the thrombin generation norm. The EXCA is not dependent on factors VIII, IX, XI and XII. Even in antithrombin III-deficient plasma, a phase of thrombin inhibition appears after the thrombin peak. Supplemented purified fibrinogen resulted in decreased thrombin generation in the important. Fibrinogen seems to act as antithrombin I; thrombin might be entrapped in the nascent fibrin. The EXCA is suitable to diagnose the level of extrinsic factors in patient plasma.

Analysis of hemostasis alterations in sepsis.

This laboratory study tested new methods to analyze hemostasis alterations in septic patients. Samples of ethylenediamine tetraacetic acid (EDTA) plasma and citrated plasma were collected from 62 patients with clinical diagnosis of sepsis. Additionally, a subset of EDTA-plasma samples from each patient was stabilized 1 + 1 with 2.5 mol/l arginine, pH 8.6, to conserve the real hemostasis activation state. EDTA-arginine plasma, EDTA plasma and citrated plasma samples were tested in duplicate. The patients at admission to the intensive care unit had 36 +/- 26 (normal, 0.8 +/- 0.2) ng/ml global endotoxin reactivity, 188 +/- 66% (normal, 100 +/- 20%) fibrinogen function, 179 +/- 66% (normal, 100 +/- 20%) fibrinogen antigen, 4.0 +/- 3.6 (normal, 0.049 +/- 0.025) microg/ml D-dimer, 313 +/- 307% (normal, 100 +/- 30%) plasmin-antiplasmin complex, 8.7 +/- 11.4 (normal, 1.1 +/- 0.7) U/ml plasminogen activator inhibitor-1, 12.1 +/- 10.5 (normal, 1.3 +/- 0.4) ng/ml thrombin-antithrombin III complex, 173 +/- 62% (normal, 100 +/- 20%) thrombin, 568 +/- 225 (normal, 140 +/- 42) pg/ml tissue factor, and 2.56 +/- 2.48 (normal, 0.19 +/- 0.04) microg/ml soluble intercellular adhesion molecule-1. Endotoxin (lipopolysaccharide and/or beta-glucan) reactivity (EDTA plasma), fibrinogen function + antigen + ratio and plasminogen activator inhibitor-1 (citrated plasma), and D-dimer, soluble intercellular adhesion molecule-1, thrombin activity (EDTA-arginine-stabilized plasma) presented large aberrations in septic patients when compared with normal values and may therefore be particularly interesting as markers of hemostasis alteration. Whether the observed alterations are of clinical significance has to be determined in well defined patient groups.

Singlet oxygen potentiates thrombolysis.

Activated polymorphonuclear neutrophils (PMN) participate in physiologic thrombolysis. PMN produce large amounts of urokinase (u-PA) and oxidants of the hypochlorite/chloramine-type that generate nonradical excited singlet oxygen ((1)O(2)). The u-PA/(1)O(2)-mediated thrombolysis was imitated in vitro. One hundred microliters microclots of normal human plasma were oxidized with 25 microL 0 to 5.0 micromoles of chloramine-T in physiol. NaCl in the absence or presence of 100 microL 6% bovine serum albumin or 100 microL normal plasma. Twenty-five microliters 0 to 167 IU/mL (related to 150 microL added supernatant) u-PA or 0 to 2.08 microg/mL t-PA were added. The absorbance at 405 nm was determined after 0 to 27 hours (37 degrees C). The specific clot turbidity was calculated, subtracting the 100% lysis absorbance from the respective measured absorbance. The chloramine-effective dose 50% (ED(50)) after 27 hours was determined in the presence of 2.6 IU/mL u-PA. The plasminogen activator-ED(25) was determined after 2 hours (37 degrees C), and the ET(25); i.e., the time needed to lyse a microclot by 25%, was determined for each respective clot-oxidation. The ED(25) of u-PA depends on the oxidation of the microclots: 1.25 micromoles chloramine/100 microL clot enhances thrombolysis approximately 20-fold; here, 25% of clot lysis is achieved within 50 minutes (using approximately 20 IU/mL u-PA), whereas approximately 5 hours are needed to lyse an unoxidized microclot by 25%. The present global assay technique imitates the u-PA/(1)O(2) aspects of physiologic thrombolysis by PMN.