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PubMed1987

Changes in fibrinolysis in the intensive care patient.

Critically ill patients have been described as having blood coagulation abnormalities that predispose to bleeding and thrombosis. We have studied plasminogen activators, alpha 2-antiplasmin, X-oligomers fibrin fragments, fibronectin, antithrombin III, fibrinogen, platelets, kaolin-cephalin clotting time and prothrombin time on admission to the intensive care unit and sequentially after 24 and 48 hours in 39 adult patients: ARDS (n = 6), trauma (n = 12), sepsis (n = 8) and a miscellanea (n = 13). A decrease in plasminogen activators associated with an increase in X-oligomers, the earliest form of cross linked fibrin degradation products, indicate that fibrin deposition and the consumption of components of fibrinolysis is a widespread condition in the ICU patients. Low fibronectin levels were related to prognosis. These findings suggest that critically ill patients must be evaluated in respect to fibrinolysis and supported when necessary with prophylactic treatment.

Adolescent↗
PubMed1987

Inhibition of human factor VIIa-tissue factor activity by placental anticoagulant protein.

Previous studies indicated that human placental anticoagulant protein, a member of the lipocortin family, prolonged the clotting time of normal plasma when clotting was induced by brain thromboplastin or by kaolin in the presence of cephalin and calcium. Using a two-stage amidolytic assay to assess factor X activation and a tritiated peptide release assay to assess factor IX activation, we have examined the ability of purified preparations of placental anticoagulant protein (Mr = 36.5 kDa) to inhibit the activation of either factor X or factor IX by a complex of human factor VIIa-tissue factor. Placental anticoagulant protein markedly inhibits factor X and factor IX activation by factor VIIa-tissue factor in a non-competitive manner with Ki values of 40 nM and 70 nM, respectively. Placental anticoagulant protein had no effect on factor Xa amidolytic activity, and its inhibitory activity was not diminished by prior incubation with antibody raised against partially purified plasma extrinsic pathway inhibitor. Binding of placental anticoagulant protein to phospholipid vesicles, crude tissue factor and purified, relipidated human brain tissue factor apoprotein was observed only in the presence of calcium ions. These results indicate that placental anticoagulant protein is a potent factor VIIa-tissue factor inhibitor and suggests that its mechanism of action involves binding to the phospholipid portion of the tissue factor lipoprotein.

Annexins↗
PubMed1993

A quantitative, semi-automated and computer-assisted test for lupus anticoagulant.

We describe the design of a quantitative test for lupus anticoagulants (LA), based on the Activated Partial Thromboplastin Time (APTT) and the Russell Viper Venom Time (RVVT). In this assay system, test plasmas mixed 1:1 with a pooled normal plasma (NP) are tested at a low as well as a high cephalin concentration, using an ACL 3000 automated clot timer. The ratio of these two clotting times, divided by the corresponding ratio for the NP, was defined as the Lupus Ratio (LR) and calculated by means of a computer program. The frequency distribution of the LR in a reference population of 150 healthy individuals was determined, and the 97.5 percentile was defined as the upper reference limit and allocated the value one Lupus Anticoagulant Unit (LA-U). Using dilutions of one strong LA positive plasma, standard curves for LA-U determination were constructed for the APTT as well as the RVVT based test, and fitted with a log-logit computer model. The sensitivity of the tests was comparable to that of the Kaolin Clotting Time (KCT). Plasma samples from warfarin treated patients were uniformly negative, while most heparin-containing plasmas were positive in both tests. Plasmas deficient in Factors V, VIII and IX were negative, whereas one Factor VIII-inhibitor containing plasma was positive in the APTT and negative in the RVVT. The present work shows that it is possible to adapt the APPT as well as the RVVT for LA quantification. With an automated clot timer and computer based calculation of results, the assays are simple and reproducible and have a high sensitivity and specificity.

Adult↗
PubMed1993

Choice of standard plasma for diagnosis and quantitation of lupus anticoagulants.

We have recently developed a computer-assisted, semi-automated test for lupus anticoagulants (LA), based on the Activated Partial Thromboplastin Time (APTT) and the Russell's Viper Venom Time (RVVT). In this test, plasma samples mixed 1:1 with pooled normal plasma are tested at one low (screening procedure) and one high (confirming procedure) cephalin concentration, using an automated clot timer. The ratio of these two clotting times, divided by the corresponding ratio for normal plasma, was defined as the Lupus Ratio (LR). Based on the LR of several dilutions of a strong LA positive plasma, a standard curve was constructed for LA quantitation in both the APTT and the RVVT system. In the present article, we discuss the use of different LA positive plasma as standards for the identification as well as quantitation of LA. Due to the heterogeneity of LA, the shape and slope of the standard curves as well as the cut-off points between "normal" and "pathological" values varied from one plasma to another. Thus, the result of LA quantitation of a given plasma varied considerably, according to the choice of standard plasma. A strong LA plasma should be chosen as a standard, since any standard plasma only allows the quantitation of plasmas with a lower LR than its own. A pool of several plasmas is also suitable as a standard. In addition to LA quantitation, such a standard could be used to define the cut-off between "normal" and "abnormal" results in each assay, as a certain dilution of the standard plasma. The present study also confirms the greater sensitivity of the APTT as compared to the RVVT for LA detection.

Adolescent↗
PubMed1995

Desmopressin (DDAVP) acts on platelets to generate platelet microparticles and enhanced procoagulant activity.

Desmopressin (DDAVP), an analog of vasopressin (AVP), has wide clinical application as an anti-hemorrhagic (AH) agent. DDAVP in vivo releases vWF from endothelial cells but is reported to have little action on platelets. However, DDAVP is often used to improve hemostasis in platelet dysfunctions. We examined the effect of DDAVP on platelet microparticle (PMP) formation and procoagulant activity in vitro using platelets from normal volunteers and in vivo in six patients receiving DDAVP therapy. In the former, platelets were incubated with DDAVP (0.5 to 25 nM) and PMP released were stained with FITC-labeled MAb alpha-GP IIb/IIIa for flow cytometry. Procoagulant activity was measured in a clot-based assay using Russel's viper venom (RVV) calibrated with cephalin. A mean increase of 2-3 fold was observed in both PMP and procoagulant activity. Parallel to these observations was a dose-dependent rise in organelle-associated Ca2+. The assays were also performed on six patients prior to and at one hour after infusion of DDAVP, and similar but lesser effects were observed. We conclude that DDAVP acts on platelets in vitro, and that these effects may contribute to the hemostatic action of DDAVP in platelet dysfunctions in vivo.

Adult↗
PubMed1984

Purification and properties of the main coagulant and anticoagulant principles of Vipera russellii snake venom.

Vipera russellii venom was separated into thirteen fractions by means of DEAE-Sephadex A-50 column chromatography. Fraction III possessed anticoagulant and phospholipase A activities and Fraction XI possessed procoagulant and caseinolytic activities, both were further purified by gel filtration on Sephacryl S-200 column. Purified procoagulant (Component II) was a two-chain protein with molecular weight of 86 000 consisting of A-chain (Mr 66 000) and B-chain (Mr 20 000). It was a glycoprotein containing 7.8% neutral sugar and 715 amino-acid residues. The procoagulant activity was 10-times that of the crude venom. It was an acidic proteinase with isoelectric point of pH 4.2. Upon heat treatment at 60 degrees C, Component II was stable at pH 5.5 and 7.2 for 3 h, but was destroyed completely after 30 min at pH 8.9. It was devoid of esterase or amidase activity. Purified anticoagulant (Component I) was a single peptide chain with molecular weight of 16 000. It was carbohydrate free and contained 136 amino-acid residues. It was a basic protein with an isoelectric point of larger than pH 10. It was a potent phospholipase A with an enzymatic activity of 510 +/- 30 mumol/min per mg using phosphatidylcholine as substrate, and 1 microgram/ml was sufficient to cause 100% hemolysis by the indirect hemolytic method. Upon heat treatment at 90 degrees C, Component I was heat stable at pH 5.5 for more than 3 h, but was destroyed completely after 2 h at pH 7.2 and 8.9. The anticoagulant activity of Component I could be neutralized by platelet factor 3, tissue thromboplastin and cephalin.

Amino Acids↗
PubMed1976

Direct phase determination for quasi-kinematical electron diffraction intensity data from organic microcrystals.

Direct phasing techniques using cosine estimates for three-phase and four-phase structure invariants in space group P1 are applied to zonal hk0 electron diffraction intensity data from an orthorhombic paraffin and a cephalin. Ab initio use of these kinematical phases is shown to derive the correct structures of the methylene subcell packings for these compounds in agreement with earlier determinations from Patterson maps.

Crystallography↗
PubMed1976

Interaction of short chain and long chain fatty acid phosphoglycerides and bile salts with prothrombin.

An equimolar mixture of phosphatidylserine and (dioleoyl) phosphatidyl-ethanolamine could substitute for brain cephalin preparations in the single stage prothrombin assay. However, no clot promoting activity was observed on the addition of any of the individual long chain fatty acid-containing phospholipids. Short chain fatty acid-containing phospholipids, such as diheptanoylphosphatidylcholine, diheptanoylphosphatidylethanolamine, diheptanoylphosphatidic acid, and dihexanoylphosphatidylcholine, or dihexanoylphosphatidylethanolamine were inhibitory under all conditions studied. Similar effects of these two general classes of phospholipids were observed in a two-stage thrombin generation system, in which a mixture of bovine Factor Xa, Factor Va, and Ca2+ were interacted with prothrombin. In the presence of 25 mM Ca2+, dioleoylphosphatidic acid or brain phosphatidylserine alone, and with other long chain phospholipids, formed complexes with bovine plasma prothrombin. On the other hand, dioleoyl-, diheptanoyl- or dihexanoylphosphatidylcholine under comparable conditions showed no binding to prothrombin. There appeared to be a small degree of binding of diheptanoylphosphatidic acid to prothrombin, but it was insufficient to cause any significant change in apparent molecular weight of prothrombin. A mixture of prothrombin, Factor V, diheptanoylphosphatidic acid/diheptanoylphosphatidylcholine and Ca2+ eluted in the void volume of Sephadex G-200, but showed a much reduced coagulant activity. Though a net negative charge on the phospholipid surface is required for phospholipid-protein interactions, this does not necessarily promote coagulant activity. Bile acids and bile salts, such as cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, lithocholic acid and dehydrocholic acid, exerted varying levels of stimulation on the prothrombin assay and thrombin generation system, but were not as effective as the phospholipids. Interestingly, no interaction of these bile acids or salts with prothrombin was noted in the presence of Ca2+. The results of these experiments suggest that negatively charged micelles per se are not sufficient for binding alone and that other chemical and physical characteristics of phospholipids are of prime importance.

Animals↗
PubMed1978

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosquamatus venom.

By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10. The anticoagulant principle possessed phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity. The anitoagulant action of the purified principle was competitively inhibited by platelet phospholid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoagulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the inactivation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.

Amino Acids↗
PubMed1987

Coagulation and fibrinolysis in post-menopausal women treated with Org OD 14.

The effects of a new synthetic steroid Org OD 14 on the haemostatic mechanism were investigated in 60 post-menopausal women, randomly allocated to 12 weeks of treatment with either 2.5 mg/day of Org OD 14 or a placebo in a double-blind, group-comparative study. Assessments were made 2 weeks before and just prior to the start of treatment, at weeks 6 and 12 during treatment, and 2 weeks after its cessation. No significant differences between the two groups were found with regard to prothrombin time, kaolin cephalin clotting time (KCCT), clotting factors VII, VIII and X, white blood count (WBC) or transaminases (ASAT, ALAT). The following statistically significant differences were seen in the Org OD 14 group: higher plasminogen, antithrombin III, haemoglobin, haematocrit and platelet count, and increased fibrinolytic activity on fibrin plates, as well as lower fibrinogen and alkaline phosphatase values. These findings indicate that Org OD 14 displays no adverse effects on coagulation, while changes in fibrinolysis seem to be beneficial for post-menopausal women.

Blood Coagulation↗
PubMed1991

A comparative study of the biological activities of rattlesnake (genera Crotalus and Sistrurus) venoms.

1. The hemorrhagic, procoagulant, anticoagulant, protease, arginine ester hydrolase, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, phospholipase A and L-amino acid oxidase activities of 50 venom samples from 20 taxa of rattlesnake (genera Crotalus and Sistrurus) were examined. 2. The results show that notwithstanding individual variations in the biological activities of Crotalus venoms and the wide ranges of certain biological activities observed, there are some common characteristics at the genus and species levels. 3. The differences in biological activities of the venoms compared can be used for differentiation of the species. Particularly useful for this purpose are the thrombin-like enzyme, protease, arginine ester hydrolase, hemorrhagic and phospholipase A activities and kaolin-cephalin clotting time measurements.

Acetylcholinesterase↗
PubMed2003

Increased calpain expression following experimental cerebral venous thrombosis in rats.

INTRODUCTION: Calpains are intracellular proteases that are activated by increased intracellular calcium with proteolytic activity mainly against the cytoskeleton. We tested the expression of calpains and their substrates in an animal model of experimental cerebral venous thrombosis. MATERIALS AND METHODS: Cerebral venous thrombosis was induced in seven male rats by rostral and caudal ligation of the superior sagittal sinus and injection of a thrombogenic cephalin suspension. Each animal survived 3 h of thrombosis. Using a polyclonal antibody against the 80 kD subunit of micro-calpain, immunohistochemistry of the region of interest (venous infarction) showed a loss of microtubule-associated protein-2. The micro-calpain-positive cells in the region of interest and normal tissue were measured using a video-imaging microscopy unit with magnification power of 400x. A cell was considered calpain positive when the nucleus and the periplasma were stained by the micro-calpain antibody. RESULTS: The mean infarct size was 13.4+/-3.7% of one whole coronal section. A total of 57+/-14% of the cells were found to be calpain positive in the region of interest, whereas 5+/-2% of all cellular elements in unaffected tissue were calpain positive (p<0.001). CONCLUSIONS: In conclusion, cerebral venous thrombosis causes an increase in calpain expression in affected tissue which is manifested by a loss of microtubule-associated protein-2. This increase might mediate secondary neuronal injury.

Animals↗
PubMed2001

Antithrombin activity of an algal polysaccharide.

In an effort to reduce the risks of a possible iatrogenic transmission of bovine spongiform encephalitis (BSE) through the use of bovine-derived medicinal products, we patented in the USA in 1999 a polysaccharide from brown algae, endowed with interesting pharmacological activities: (a) concentration-dependent inhibition of thromboplastin or cephalin-kaolin-induced thrombin generation from platelets, (b) concentration-dependent inhibition of thrombin-induced platelet aggregation, (c) thrombin has hypotensive effect, which was blunted and zeroed by our fucansulfate in a dose-dependent way, (d) when aortae are stimulated with thrombin, they become stickier for polymorphonucleated leukocytes (PMNs); our fucansulfate decreased concentration-dependently, PMNs sticking to autologous rabbit aortae, (e) dose-dependent inhibition of thrombin-induced thrombosis. All the above data suggest that our fucansulfate could be a heparin substitute endowed with antithrombotic and anti-inflammatory activities, devoid or the problems caused to heparin by its animal origin, i.e., possible prion protein contamination.

Animals↗
PubMed1976

Measuring partial thromboplastin-time. An international collaborative study.

A series of collaborative exercises on the partial thromboplastin-time (P.T.T.) test, involving over three hundred hospital centres in Britain and overseas, were performed in 1975. Lyophilised test plasmas were issued from the World Health Organisation Collaborating Centre for Anticoagulant Control Reagents to participants, together with a standardised reference P.T.T. reagent and a standard technique. Hospitals were asked to test the plasma samples with the standardised reagent and technique in parallel with their customary local P.T.T. reagent and method. The overall success-rate in detecting the intrinsic clotting abnormality in the eight abnormal test samples was higher with the standardised reagent and technique than with all other reagents. Furthermore, fewer hospitals obtained false positive results when the normal plasma sample was tested with the standardised method rather than with their usual routine reagents. An index was used to measure the success-rate of the P.T.T. reagents in correctly identifying the test plasmas as normal or abnormal. The eight test plasmas showed a varying degree of abnormality. A system of "weighting" was therefore introduced as the failure of a P.T.T. method to detect more severe defects was regarded as more serious. Although hospitals were unfamiliar with the standardised method, the results established its superiority over all other P.T.T. reagents included in the trials in sufficient numbers for analysis. Failures with commercial reagents may have been caused by insensitivity of the cephalin extracts or the unreliability of the manufacturers recommended techniques. Since the same laboratories obtained good results with the standardised method technical failure can be excluded.

Blood Coagulation Disorders↗
PubMed1977

A heparin analogue with specific action on antithrombin III.

The effects of heparin and a semi-synthetic heparin analogue were compared in vivo and in vitro. The two drugs differed strikingly in their in-vitro behaviour: unlike heparin, the heparin analogue had little effect in a specific heparin assay or on overall clotting, as measured by the kaolin-cephalin clotting time (K.C.C.T.). When given by parenteral injection, the heparin analogue had almost the same potentiating effect on antithrombin III as mucous heparin, but without a comparable effect on the K.C.C.T. These observations suggest that the heparin analogue may have desirable characteristics for the prophylaxis of venous thrombosis, since it selectively potentiates antithrombin III in vivo while having little effect on overall clotting.

Adult↗
PubMed1997

Dermatophilus congolensis: strain differences in expression of phospholipase activities.

Interactions between Dermatophilus congolensis strains and with other bacteria of known haemolytic activities were used to elucidate the complex nature of haemolytic activities present in various D. congolensis strains. This was further analysed by measuring their specific phospholipase activities against defined substrates by thin layer chromatography. D. congolensis strains demonstrated haemolytic interactions (synergistic or antagonistic) with other D. congolensis strains and also other species of bacteria. Most isolates expressed lyso-phospholipase-D activity, while various strains also expressed sphingomyelinase-D activity, phospholipase-A versus phosphatidylcholines and/or cephalins, phospholipase-D versus phosphatidylcholines or all these activities, under the culture conditions used.

Actinomycetales↗
PubMed2003

No acute effect of red wine on the coagulation pathway in healthy men.

Binge drinking is associated with an increased risk of cardiovascular events. Those events often happen within hours after alcohol is consumed. Apart from arrhythmias and changes in blood pressure, these events may be caused by an acute (i.e., occurring within a 24-h period) shift of the hemostatic balance in a thrombogenic direction. Alcohol can influence platelet aggregation and inhibit fibrinolysis, but little is known about its direct effect on coagulation. In the current study, parameters of coagulation, reflecting either stimulation or inhibition, were measured 5 and 15 h after the consumption of four (62.5 g of alcohol) and eight (125 g of alcohol) glasses of red wine. Both doses had no direct effect on activated cephalin time, thrombin-antithrombin complexes, factors VII and VIII, and von Willebrand factor. In contrast with the observed effects on thrombocytes and fibrinolysis, the consumption of large amounts of wine does not influence the coagulation pathway.

Adult↗
PubMed1992

[Efficacy and tolerance of Elohes 6% during peroperative normovolemic dilution].

This study assessed the cardiovascular stability of intraoperative normovolaemic haemodilution carried out with 6% Elohes during the exchange, the intraoperative, recovery and postoperative periods, as well as its effects on coagulation and renal function. Ten patients, ranked ASA 1, were included. Patients were premedicated with 100 mg of hydroxyzine, and anaesthesia was induced with 2 mg.kg-1 propofol, 3 micrograms.kg-1 fentanyl, and 0.5 mg.kg-1 atracurium. Maintenance was obtained with propofol, fentanyl and 50% nitrous oxide in oxygen. Haemodilution, carried out with a peripheral venous catheter, size 14 G, consisted in the removal of 15 ml.kg-1 of blood and its simultaneous replacement with Elohes 6%. Heart rate, systolic, diastolic and mean blood pressures were recorded before anaesthetic induction, every 5 min during the haemodilution, and thereafter during surgery, recovery and postoperative periods, up to the third postoperative day. Blood haemoglobin, fibrinogen, prothrombin, sodium, potassium, urea and creatinine concentrations, haematocrit, platelet count, and bleeding and activated cephalin times were assessed before and immediately after haemodilution, and on postoperative days 1 and 3. A mean of 1,295 +/- 68 ml of blood were removed during a 32 +/- 2 min period, and replaced by 1,315 +/- 64 ml of Elohes. Haematocrit decreased from 44 +/- 1.1% to 29.7 +/- 0.8%. There were no significant alterations in other parameters, except for an increase in heart rate at the time of extubation. Cardiovascular parameters, as well as the haemodilution, were stable up to the end of the study period. It is concluded that replacing blood with Elohes for intentional haemodilution affords convenient cardiovascular stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗