bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparin Prevents Stasis-Induced Thrombosis through Protection of the Venous Endothelial Cells: An Electron Microscopic Study in Rabbits
Authors: Trabucchi E. , Foschi D. , Marazzi M. , Abelli P. , Andriuoli G. , Lami F. , Scagnol I. , Del Soldato P. , De Santis F. , Montorsi W.
Published: April 2009

This study was performed to see whether or not protection of the endothelial cells contributes to the antithrombotic effects of heparin. New Zealand albino rabbits were subjected to jugular vein stasis by single caudal ligation for 2 h. Three treatments were given: saline (control group), heparin (0.2 mg/kg) 5 or 45 min before ligature of the vein. Groups of 6–8 animals were killed at 0, 5, 15, 30 and 120 min. The following parameters were determined: (1) involution and damage of the endothelial cells by scanning and transmission electron microscopy; (2) incidence and weight of thrombi in the lumens of the veins after 2 h stasis, and (3) effects of heparin on APTT and anti-Xa activity. In the control group, stasis caused a considerable involution of the endothelial cells in the first 30 min, followed by fibrin deposition and thrombus generation. Heparin strongly reduced the damage to the endothelial cells, with very evident protection of the cell membranes, and prevented thrombus generation: there were significant decreases in both incidence and weight of thrombi. These effects of heparin were evident both shortly after (maximal anticoagulant effect) and long after (no anticoagulant effect) pretreatment. We think that, under the experimental conditions we used, heparin prevented venous thrombosis at least partially by protection of the endothelial cells, through unknown mechanisms.
Coagulation and Fibrinolysis in Orthotopic Liver Transplantation: Role of the Recipient’s Disease and Use of Antithrombin III Concentrates
Authors: Palareti G. , Legnani C. , Maccaferri M. , Gozzetti G. , Mazziotti A. , Martinelli G. , Zanello M. , Sama C. , Coccheri S.
Published: April 2009

In this study we report the data obtained from extensive haemostatic testing of 25 patients undergoing orthotopic liver transplantation and the results of an open randomized pilot trial of antithrombin III concentrate administration during surgery. Marked differences in transfusional needs and in pre- and intraoperative blood coagulation and fibrinolytic changes were observed between recipients with liver cirrhosis and those with primary biliary cirrhosis. In the former, the increases in tissue-type plasminogen activator activity, total euglobulin fibrinolytic activity, and fibrin-derived degradation products occurred earlier and were more marked, as were the signs of increased thrombin formation. Supplementation of antithrombin III concentrate during surgery failed to induce significant changes in the main parameters studied and in the transfusional needs.
Effects of the Synthetic Ether Phospholipid KO-286011 on Cells and Components of Rabbit Blood
Authors: Hofmann Barbara , Greiner Jens , Glusa Erika , Kertscher Heinz-Peter , Ostermann Gunter
Published: April 2009

The ether phospholipid KO-286011, which is related to platelet-activating factor (PAF), was examined for PAF-antagonistic action on rabbit platelets both in vitro and ex vivo. It inhibited the aggregation of washed platelets induced by PAF (5 nmol/l) concentration dependently (IC50 = 6.5 × 10-7 mol/l). After injecting 0.5 mg/kg to rabbits, the ex vivo PAF-mediated aggregation in heparinized platelet-rich plasma was influenced selectively. The inhibition was short and maximal after 5 min. In contrast, hematocrit, counts of peripheral platelets and leukocytes, hemoglobin content, partial thromboplastin time, prothrombin time, and fibrinolytic activity in plasma were not affected. However, the therapeutic range of KO-286011 is limited because of membranolytic activity at higher dosages, leading to hemolysis and drop of circulating platelet counts.
Comparative Study on the Antithrombotic Efficacy of Four Low-Molecular-Weight Heparins in Three Different Models of Experimental Venous Thrombosis
Authors: Monreal Manuel , Silveira Pierre , Monreal Luis , Monasterio Jasone , Angles Anna María , Lafoz Elena , Lorente Laura
Published: April 2009

In a randomized, blind study, both the antithrombotic efficacy (reduction of thrombus weight) and potency (anti-Xa activity) of several commercially available low-molecular-weight heparins (LMWHs) were compared with those of unfractioned heparin (UFH) and placebo. Three different thrombogenic challenges were used: venous thrombosis was induced by direct endothelial damage in 60 New Zealand rabbits (group I), intracarotid injection of bovine thrombin in an additional series of 60 rabbits (group II), or after inferior-vena-cava ligature in 60 Wistar rats (group III). The drugs were administered subcutaneously 2 h before surgery in a blind fashion. The doses recommended for clinical practice were used (adjusted by body weight), except in group II animals, in whom doses were doubled. No differences were found between UFH and most LMWHs in terms of reduction of thrombus weight in group I animals. But UFH showed a weaker antithrombotic efficacy in the other two models. Similarly, one of the LMWHs used (Clexane) proved to be not as effective as the remainder. However, only clinical studies will provide enough information to verify these differences. Additionally, our findings confirm that the antithrombotic efficacy of a given drug differs according to the stimulus used to induce venous thrombosis.
In vitro Inhibition by Defibrotide of Monocyte Superoxide Anion Generation: A Possible Mechanism for the Antithrombotic Effect of a Polydeoxyribonucleotide-Derived Drug
Authors: Cirillo Ferdinando , Margaglione Maurizio , Vecchione Gennaro , Ames Paul R.J. , Coppola Antonio , Grandone Elvira , Cerbone Anna Maria , Marelli Claudio , Di Minno Giovanni
Published: April 2009

In an attempt to elucidate the antithrombotic potential of defibrotide (D) we have evaluated several functions of monocytes from 7 healthy subjects before and after in vitro incubation of the cells with increasing concentrations of this drug. At concentrations as high as 40 μg/ml, D hardly affected the expression of both the procoagulant activity of monocytes and the formation of superoxide anion in response to 1 mg/ml zymosan (STZ). In contrast, at concentrations that may be achieved in vivo following the administration of the drug (5–20 μg/ml), D impaired in a dose-dependent manner (p < 0.05) the generation of O-2 in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 μM) or calcium ionophore A23187 (10 μM). Regardless of the agonist employed, at concentrations between 1 and 5 mM, extracellular Ca2+ had little effect on the impairment of superoxide anion generation by D. In contrast, the inhibitory effect was time-dependent, the maximum impairment ( > 30%) being observed when the cells were preincubated with the drug for 20 h. These data support the concept that the antithrombotic potential of D involves the ability of the drug to affect the generation of free radicals by leukocytes and suggest that future in vivo studies for the evaluation of the activity of D should take into account the role of monocytes in hemostasis and thrombosis.
Comparison of the Appropriateness of the Latex and Elisa Plasma D-Dimer Determination for the Diagnosis of Deep Venous Thrombosis
Authors: Chang-Liem G.S. , Lustermans F.A.Th. , van Wersch J.W.J.
Published: April 2009

Thirty-two patients suspected of deep venous thrombosis (DVT) of the leg were evaluated after simple plasma tests (latex D-dimer and Elisa D-dimer) against echography and phlebography as the gold standard of DVT. Seven patients showed negative results in the latter test. The classification conformity between the D-dimer methods amounted 84.4%. With regard to the diagnostic classification a high specificity of 100% was found for the latex D-dimer test at the cut-off level of 450 ng/ml, whereas for sensitivity, 96% was reached. The Elisa D-dimer test was striking by its high sensitivity of 100%, but its low specificity of 28.5 %. In summary, these results demonstrate that when there is a suspicion of DVT according to the clinical symptoms, a negative latex test may reject the diagnosis, whereas the latex test is less useful to establish the diagnosis. On the contrary, the Elisa .D-dimer test has a too high false-positive rate. The latex D-dimer test seems to be a reliable tool to exclude the diagnosis after which an ascending contrast venography can be omitted.
Degradation of Human Factor X by Human Polymorphonuclear Leucocyte Cathepsin G and Elastase
Authors: Turkington P.T.
Published: April 2009

Cathepsin G and elastase from human polymorphonuclear leucocytes were used in vitro to digest human factor X. Clotting assays showed that both proteinases affected a rapid loss in the coagulant activity of factor X. Calcium ions almost totally protected the coagulant activity of factor X against the action of cathepsin G but not elastase. Polyacrylamide gel electrophoresis (in nonreducing conditions and in the presence of SDS) indicated that the proteolytic action of cathepsin G led to the removal of a peptide of low molecular mass (pX) with the consequent formation of a single stable high molecular mass product (PX). SDS electrophoresis (under reducing conditions and in the presence of SDS) indicated that a pX was derived from the light chain of factor X. The proteolytic action of elastase led to the formation of numerous degradation products. Analysis of the products generated by the action of cathepsin G indicated that cathepsin G cleaved position Phe40:Trp41 in the light chain of factor X. In the presence of citrated plasma, cathepsin G but not elastase, was responsible for a loss in coagulant activity.
Studies of von Willebrand Factor in Essential Thrombocythemia Patients Treated with Alpha-2b Recombinant Interferon
Authors: Mazzucconi M.G. , Ferrari A. , Solinas S. , Vitale A. , Chistolini A. , Federici A.B. , Mandelli F.
Published: April 2009

The crucial role of the von Willebrand Factor (vWF) and its interaction with platelets in myeloproliferative disorders (MPD) have emerged in recent years. Recently, many authors have reported the therapeutical efficacy of interferon (IFN) in MPD with thrombocytosis in decreasing platelet number. The purpose of our report is to study the modifications of vWF in a series of 20 patients affected by essential thrombocythemia (ET) or MPD with thrombocytosis, treated with α2b recombinant IFN (α2b -rlFN). Patients were studied before treatment and after complete or partial response: vWF-related properties, bleeding time (BT) and ristocetin-induced platelet aggregation (RIPA) were evaluated. Before treatment, we found prolonged BT in 5 patients (25%), abnormal RIPA in 8 (40%), reduced factor VIII coagulant activity (VIIl·C) in 2 (10%), reduced vWF-related antigen (vWF:Ag) in 5 (25%) and low vWF:ristocetin cofactor (vWF:Ricof) in 5 (25%). Twelve subjects were evaluated after hematologic remission: in all patients, BT, VIII:C, vWF:Ag and vWF:Ricof were within normal range or upper normal limits. RIPA was abnormal in 7 subjects. Multimer patterns of vWF were performed in 3 patients before and after treatment: 2 of them showed loss of high-molecular-weight multimers that seemed to recover at remission. IFN seems to induce improvement of platelet number and their functions in MPD with thrombocytosis.
Incomplete Reversal of Enoxaparin-Induced Bleeding by Protamine Sulfate
Authors: Bang Christen J. , Berstad Arnold , Talstad Ingebrigt
Published: April 2009

The neutralizing effect of protamine sulfate on enoxaparin-induced bleeding was compared in two rat models: one employing the gastric mucosa, and the other the tail skin, using a 2:1 ratio of protamine sulfate to enoxaparin on a weight basis. Whereas protamine sulfate reduced the median bleeding time (from 20 to 9.5 min) and blood loss (80%) in the gastric mucosa, an apparent ‘all-or-none’ response was seen in the tail skin, in agreement with a different hemostatic mechanism in the two bleeding models. In both models, protamine sulfate incompletely reversed the bleeding induced by enoxaparin.
Simulation Model for Thrombin Generation in Plasma
Authors: Willems George M. , Lindhout Theo , Hermens Wim Th. , Hemker Coenraad
Published: April 2009

A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1–10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.
Interaction of Feedback Control and Product Inhibition in the Activation of Factor X by Factors IXa and VIII
Authors: Jesty Jolyon
Published: April 2009

A simple numerical model of the activation of factor X by factors IXa and VIII has been constructed in order to identify and examine the major controls that operate in a nonflowing system in the presence of (1) inhibitors of factor Xa and (2) feedback activation of factor VIII by factor Xa. The model confirms, and allows parameter estimation for, (1) the control of factor Xa yield by factor Villa decay; (2) the control of generation-curve area by the rate of factor Xa inhibition; and (3) the reduction in the factor Villa decay rate in the presence of factor IXa. Beyond confirmation of existing data, the model also predicts that below a definite, but very low, threshold level of factor IXa (≤ 10 pM), minimal feedback activation of factor VIII will occur. The concentration of factor IXa at which the threshold is observed in simulations is dependent on the rate of inhibition of factor Xa.
Extrinsic Pathway Inhibitor – The Key to Feedback Control of Blood Coagulation Initiated by Tissue Thromboplastin
Authors: Sandset Per Morten , Abildgaard Ulrich
Published: April 2009

Extrinsic pathway inhibitor (EPI) is a Kunitz type serine protease inhibitor. EPI is a potent inhibitor of the factor VIIa/thromboplastin (TP) complex in the presence of factor Xa and is also a direct inhibitor of factor Xa. The inhibitory mechanism is complex and is currently thought to involve, in a first step, the formation of a EPI-factor Xa complex, and, in a second step, the formation a quaternary EPI-factor Xa-factor VIIa-TP complex. In the blood vessels, EPI is confined to three different pools. A major pool of EPI is bound to the endothelial surface, and this fraction may be released by heparin. Plasma contains a second, but smaller pool of EPI (≈ 10–50% of the endothelial surface pool) at a concentration of 50–100ng/ml. This pool consists mostly of EPI-lipoprotein complexes and only less than 10% is carrier-free EPI. A third pool of EPI is confined to platelets ( < 10% of the plasma pool). The biological role of these pools has not yet been clarified, but some evidence suggest that the carrier-free EPI is biologically most active. In patients, disseminated intravascular coagulation may continue despite normal or even elevated EPI levels. However, evidence has now been provided to indicate that EPI can inhibit factor VIIa/TP complexes formed in vivo to prevent the effect of limited amounts of TP. Taken together, the present knowledge of EPI indicates that EPI functions as a key inhibitor to feedback control of blood coagulation initiated by TP.
Modulation of the Enzymatic Activity of α-Thrombin by Polyanions: Consequences on Intrinsic Activation of Factor V and Factor VIII
Authors: Ofosu F.A.
Published: April 2009

The polyanions heparin and dermatan sulfate catalyze α-thrombin inhibition and can delay the onset of factor VIII and factor V necessary for intrinsic prothrombin activation to begin in plasma. These polyanions bind α-thrombin at its anion-binding exosite(s), structural domain(s) occupancy of which may alter the properties of the fibrin(ogen) recognition exosite of α-thrombin. We compared how such four polyanions influenced factor VIII and factor V activation during intrinsic coagulation. A pentasaccharide with high affinity for antithrombin III and the C-terminal docedapeptide fragment of hirudin (hirugen) which occupy the anion-binding and fibrin(ogen) recognition exosites of α-thrombin, respectively, could not significantly inhibit factor VIII and factor V activation. In contrast, heparin and a bis-lactobionic acid, both of which catalyzed α-thrombin inhibition, could effectively inhibit factor VIII and factor V activation. These results suggest that occupancy of fibrin(ogen) or anionbinding exosites by itself does not provide a necessary and sufficient condition for catalysis of thrombin inhibition or the inhibition thrombin-mediated amplification reactions.
Linearized Model for the Initiation of Factor Va, and Thrombin Generation
Authors: Willems George M.
Published: April 2009

A simple model of the initiation of thrombin formation in plasma as a response to factor Xa generation was constructed. In this model factor Xa is considered as an input with a constant concentration. Substrate depletion and inactivation by activated protein C are neglected. The resulting linear model allows a closed form solution by standard methods. With values of the reaction rate constants, as determined in purified systems, this model predicts a highly explosive and complete activation of factor V and prothrombin as a response to any given (steady state) factor Xa concentration even in situations where prothrombinase and(/or) thrombin are rapidly inactivated. However, the time delay to rapid thrombin production becomes longer at lower factor Xa concentrations. Analysis of this time delay as a function of the factor Xa concentration indicates that the gain of the feedback loop of factor V activation by thrombin is so high that the contribution of factor V activation by factor Xa is relatively unimportant for factor Xa concentrations in the nanomolar range. It appears that the time lag is mainly determined by the gain of this feedback loop: similar proportional reductions of each of these reaction rates causes a similar effect. The effects of moderately enhanced inhibition rates of thrombin and prothrombinase on the time delay depend strongly on factor Xa concentration. Only a minor prolongation of the delay is predicted for factor Xa concentrations in the nanomolar range, but for factor Xa concentrations in the 1–10 pM range, the enhanced decay will cause considerable delays. Simultaneous reduction of the turnover rate of prothrombinase results in much larger delays for the entire range of factor Xa concentrations.
Heparin Requires both Antithrombin and Extrinsic Pathway Inhibitor for Its Anticoagulant Effect in Human Blood
Authors: Abildgaard Ulrich , Lindahl Anne Karin , Sandset Per Morten
Published: April 2009

Heparinization of blood inhibited the generation of fibrinopeptide A (FPA) after addition of thromboplastin (TP). Heparinization was more effective when performed in vivo than in vitro; the amounts of FPA at 60 s incubation were 8% and 32%, respectively, of control values in nonheparinized blood. When monospecific, neutralizing IgG against extrinsic pathway inhibitor (anti-EPI) were added to heparinized blood prior to TP, the amount of FPA increased to 65%. When monospecific IgG blocking antithrombin (anti-AT) was used, the amount of FPA increased to values similar to those in nonheparinized blood. When anti-AT and anti-EPI were both added to heparinized blood, FPA was generated about 25% faster than in normal blood. These results show that EPI contributes significantly to the anticoagulant effect of heparin in human blood.
Location of the Quinine-Dependent Epitope on Platelet Glycoprotein IlIa
Authors: Pfueller Sharron L. , Cortizo Fernando , Gibson Jannine , Bilston Robyn , Sloan Ian G.
Published: April 2009

Quinine-dependent (Q) IgG antibodies (Q.Ab) in drug-induced immune thrombocytopenia are heterogeneous and bind to different platelet surface glycoproteins (GP), namely GPIb, IX, lIb, IlIa and an unidentified 57-kDa membrane proteins. Although both the Q-dependent epitope on GPIIIa and the P1A1 antigen require intact disulphide bonds for their expression, they are distinct because Q.Ab bind to GPIIIa lacking P1A1. Epitopes for both antigens were examined on Western blots of either intact washed human platelets or purified GPIIIa. When intact platelets were digested with trypsin and washed and solubilised prior to electrophoresis, membrane-associated fragments of GPIIIa of 78 kDa were found to be reactive with both antibodies. In addition, 60- and 68-kDa fragments bound anti-P1A1 but not Q.Ab. Similar digestion with chymotrypsin produced only 60-kDa fragments containing both epitopes. Digestion of purified GPIIIa with chymotrypsin produced 60-kDa peptides reactive with Q.Ab and anti-P1A1 in immunoblotting studies. Similar digestion with elastase produced 58-kDa fragments also containing the epitopes for both antibodies. Longer digestion times or sequential digestion with different enzymes did not reveal extra fragments. However, immunoprecipitation of trypsin-digested 125I-labelled GPIIIa with affinity-purified Q.Ab produced a 17-kDa fragment containing the Q-dependent epitope.
Sex-Related Differences in Plasminogen Activator Activity and Plasminogen Activator Inhibition of Human and Animal Kidneys: Effect of Orchidectomy or Ovariectomy
Authors: Smokovitis A. , Kokolis N. , Taitzoglou I.
Published: April 2009

Plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) have been studied with spectrophotometric methods in extracts of human, bovine, ovine and rat kidneys of both sexes. In all species studied, renal PAA (cortex or medulla) was higher in females than in males. The PAA was also higher in the medulla than in the cortex in all species and both sexes. The PAA was due to both types of plasminogen activator; tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). In the human kidney (cortex or medulla) the measurement of t-PA antigen showed that t-PA is higher in females than in males; t-PA is also higher in the medulla than in the cortex in both sexes. The PAI showed the opposite pattern in all species studied; it was lower in females than in males. It was also lower in the medulla than in the cortex. PAI-1 was identified in the human kidney. Sex-related differences in renal PAA or PAI almost disappeared after bilateral orchidectomy in rats. PI showed no sex or regional differences in the species studied. Sex-related differences in renal PAA and PAI in man and various animal species might be of physiological or pathophysiological importance.
Simulation of the Extrinsic Pathway of the Plasmatic Clotting System
Authors: Baumann Peter , Heuck Claus-C.
Published: April 2009

Activation of the extrinsic plasmatic clotting system was simulated in a computerized analysis. The results were compared with previously described experimental investigations in plasma with isolated clotting factor deficiency, which led to the conclusion that the sequence of patterns for constants of a function describing the extinction curves is related to the sequence of steps of clotting factor activation. The kinetics of activation resulting in extinction curves that correspond to the curves obtained from experimental measurements are described by sets of stiff coupled linear differential equations. The set of functions can be numerically solved without further approximations. As for experimental extinction curves the simulated extinction curves are characterized by an empirical function with three constants. The distribution patterns for the constants are qualitatively similar to experimental patterns, if the following assumptions are made: (1) feedback reaction occurs from factor Ila via factor V, and (2) the conversion of factor II by factor Xa occurs at a rate considerably slower than the conversion of factor II by the prothrombinase complex. A feedback reaction by factor Xa via factor VII accelerates the formation of factor X, although the distribution pattern remains similar to the distribution pattern for a mechanism without the feedback reaction, provided that the initial activation of factor X occurs at a fast rate. A feedback reaction by factor Xa via factor V in addition to the feedback reaction by factor Ila via factor V accelerates the activation, while the pattern distribution remains unchanged. The simultaneous inhibition of factor Xa and factor Ila by antithrombin III does not change the pattern distribution, while the formation of activated factor II is decelerated.
Determination of Leukocyte Elastase Concentration in Plasma and Serum by a Simple Method Using a Specific Synthetic Substrate
Authors: Nagamatsu Y. , Yamamoto J. , Fukudd A. , Ohta M. , Tsuda Y. , Okada Y.
Published: April 2009

The concentration of leukocyte elastase (ELP) in plasma and serum was determined by an amidolysis method using a specific synthetic substrate for ELP, Suc-Ala-Tyr-Leu-Val-pNA. Results were compared with those using ELISA. ELP levels in plasma from healthy donors were similar to those determined by ELISA; however, the levels in serum were lower than those determined by ELISA. Correlation coefficients of ELP levels in plasma and serum as measured by the two methods were 0.75 (amidolysis) and 0.90 (ELISA). On the other hand, the correlation coefficient between serum ELP by the two methods was 0.83. Half of the ELP levels in plasma from 150 patients and serum from 400 patients were significantly elevated when compared with those from healthy donors, and the ELP elevation determined by amidolytic assay correlated with some variables in blood, namely fibrin(ogen)-degraded products, fibrinogen, GOT, GPT, γ-GTP, LDH and leucine aminopeptidase. Despite the fact that the amidolysis method detects the α2-macroglobulin-ELP complex while ELISA detects the α1-antitrypsin-ELP complex, a comparative study showed amidolysis to provide sufficiently sensitive measurement of both plasma and serum ELP.
Antiplatelet Glycoprotein lb Monoclonal Antibody (OP-Fl) Totally Abolishes Ristocetin-Induced von Willebrand Factor Binding, but Has Minimal Effect on the Botrocetin-Induced Binding
Authors: Nishio Kenji , Fujimura Yoshihiro , Nishida Sachiyo , Takeda Ichiro , Yoshioka Akira , Fukui Hiromu , Tomiyama Yoshiaki , Kurata Yoshiyuki
Published: April 2009

We describe here a new antiplatelet glycoprotein (GP) lb monoclonal antibody (MoAb) designated OP-Fl (IgGlκ). Both OP-Fl and a well-characterized anti-GPIb MoAb, AP-1, totally abolished ristocetin-induced von Willebrand factor (vWF) binding to platelets and desialylated vWF binding to platelets at an IgG concentration of 2–8 μg/ml. AP-1 also blocked snake venom botrocetin-induced vWF binding at a similar IgG concentration, whereas OP-Fl had a minimal effect on botrocetin-induced binding. At a higher IgG concentration (150 μg/ml), OP-Fl inhibited botrocetin-induced binding by 50%. AP-1 (IgG) did not interfere with binding of [125I]OP-F1 (IgG) to platelets. Thus, the epitope involved in the binding of OP-Fl or AP-1 appears to be quite different. These results suggest that the vWF binding site(s) on the GPIb molecule generated by these inducers is in close proximity but not completely identical.
Possible Involvement of Platelets in Plasmodium yoelii nigeriensis Malaria Infection in Mice
Authors: Oyekan A.O. , Onabanjo A.O.
Published: April 2009

The possible involvement of platelets in the pathogenesis of malaria was examined by monitoring the changes in platelet function (bleeding time [BT] and thrombin time [TT]) concomitantly with changes in packed cell volume (PCV) and erythrocyte infection rate (EIR) in Plasmodium yoelii nigeriensis infection in mice. In untreated plasmodium-infected mice, there was a progressive reduction (day 1–7) in BT from 120.0 ± 12.6 s to 77.5 ± 5.9 s (p < 0.005), a reduction in TT from 26. 8 ± 1.2 s to 17.8 ± 1.2 s (p < 0.005), an increase in EIR from 0 to 64.6 ± 6.3% and a reduction in PCV from 44.9 ± 1.9% to 21.5 ± 3.9% (p < 0.001). Mortality on day 9 was 100%. Antiplatelet serum treatment of plasmodium-infected mice protected against malaria and there was a blunting of the malaria-induced changes in platelet function, EIR and PCV. The values obtained in these rats were significantly higher than those of the untreated malarious mice. The respective values obtained on day 1 were comparable to those of control mice. Data obtained on day 7 were: BT, 106.4 ± 7.4 s (p < 0.05), TT, 22.7 ± 1.1 s(p < 0.05), EIR, 45.7 ± 3.2% (p < 0.01) and PCV, 39.5 ± 2.0% (p < 0.01). Mortality on day 9 of infection was 60%. The protection by antiserum was not due to its thrombocytopenic response as busulphan-induced thrombocytopenia did not have the same effect. These results suggest that platelets play an important role in malaria infection as well as in the attendant coagulopathic complications.
The History of Leeching and Hirudin
Authors: Fields William S.
Published: April 2009

Leeching is an art dating back at least to ancient Egypt. It reached its zenith in the late 18th and early 19th centuries. The antithrombotic quality of leech saliva was first noted by Haycraft in 1884 and the active anticoagulant ingredient isolated in 1904 by Jacoby. He gave this agent the name ‘hirudin’. Hirudin was isolated in pure crystalline form by Markwardt in 1957 and first produced in quantity by genetic engineering in 1986. The salivary glands of several species of leech also contain other biologically active substances which are currently undergoing investigation.
Effect of Hirudin on the Chemotactic Properties of Alpha-Thrombin
Authors: Morin A. , Marchand-Arvier M. , Doutremepuich C. , Vigneron C.
Published: April 2009

A study by an agarose technique of the chemotactic power of human α-thrombin on polymorphonuclear leukocytes reveals that this enzyme has a chemotactic activity. Hirudin, which inhibits the coagulant properties of thrombin, suppresses these chemotactic properties. This effect could be explained by the binding of hirudin to the structural domain of α-thrombin involved in its chemotactic activity.
A Fast Photometric Assay for the Determination of Hirudin
Authors: Spannagl M. , Bichler H. , Lill H. , Schramm W.
Published: April 2009

Recombinant hirudin, a potent and specific thrombin inhibitor, is considered for anticoagulant therapy. Therefore we developed a fast and sensitive chromogenic assay for its determination in plasma. Samples can be assayed directly if aprotinin, Polybrene® and urea are added to the reagent mixture. The influence of progressive inhibitors is excluded by a short incubation time. The samples (20 μl) are diluted with 1 ml reagent mixture (0.2 MTris, 0.025 MNaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitory units/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH units/ml bovine thrombin). After an incubation time of 1 min, 100 μl Chromoyzm TH® (Tos-Gly-Pro-Arg-pNA, 1.9 mM) is added. Δ absorbance/min is linear at least up to 3 min. The calibration curve is linear up to at least 800 ng hirudin/ml plasma. The inter- and intraassay coefficient of variation in hirudin spiked normal plasma is below 5%. The detection limit is at 25 ng hirudin/ml. In plasma samples obtained from healthy subjects under hirudin therapy, a good correlation was found to the activated partial thromboplastin time (r = 0.89). In conclusion, we describe a fast and simple chromogenic substrate test to assay hirudin in plasma. Under assay conditions, the influence of endogenous thrombin inhibitors can be neglected.
Laboratory Assays for the Evaluation of Recombinant Hirudin
Authors: Walenga Jeanine M. , Hoppensteadt Debra , Koza Michael , Wallock Michael , Pifarre Roque , Fareed Jawed
Published: April 2009

Several laboratory methods are available to measure the anticoagulant activity of recombinant hirudin (r-hirudin), a potent thrombin inhibitor. These assays include clot-based, amidolytic, immunologic and physicochemical techniques. Although r-hirudin, like heparin, is an effective anticoagulant, the mechanism of action of the two agents is different. Thus it is not surprising that the global tests, such as the prothrombin time (PT), partial thromboplastin time (APTT) and the Heptest® (Haemachem, Inc., St. Louis, Mo., USA), do not show adequate responses to r-hirudin. In the range of 0.5–10.0 μg/ml, where full anti-coagulation is achieved, as determined by animal models of thrombosis, these assays show little to no prolongation of the time to clot. In order to find a more suitable assay system, modifications of the above assays were evaluated. The diluted APTT and diluted Heptest showed linear concentration-dependent responses to lower levels of r-hirudin with an enhanced sensitivity than that of the classical assays. On the other hand, the diluted thrombin time was too sensitive. Whole-blood clotting assays, ACT® and thrombelastograph, effectively measured r-hirudin levels up to 25 μg/ml. The amidolytic anti-factor IIa assay, specific for evaluating direct thrombin inhibition, was very effective particularly when modified to decrease the sample:thrombin ratio. This assay may be useful in quality control since it is biochemically defined, and reagents are easily standardized. The relevance of the results of the anti-IIa assay to clinical conditions, however, remains to be determined. Thrombin generation assays have limited value in monitoring the anticoagulant effect of r-hirudin since the effect of thrombin inhibition by r-hirudin on coagulation feedback mechanisms, and thus the effect on thrombin generation, appears to be minimal. Immunologic methods such as ELISA and RIA are under development, but they may only be useful for the direct quantitation of absolute levels of r-hirudin and not for monitoring the clinical anticoagulant action. Furthermore, these assays are only sensitive to sub-μg/ml levels. Therefore, thrombin-based clotting and amidolytic assays may at present be the best choice for evaluating the functional, clinical antithrombotic effects of r-hirudin.
The Effects of Recombinant Desulphatohirudin on Arterial Thrombosis in Rats
Authors: Talbot Mark D. , Ambler John , Butler Keith D. , Lees Christopher M. , Mitchell Kevin A. , Peters Robin F. , Tweed Morris F. , Wallis Robert B.
Published: April 2009

The role of thrombin in the formation of arterial thrombi is poorly understood. With the new availability of the specific thrombin inhibitor, recombinant desulphatohirudin, in large quantities this is now under investigation. A new model of arterial thrombosis in rats is described where a thrombus is formed on a mechanically injured vessel in vivo. Both platelet and fibrin deposition is inhibited by a recombinant hirudin (CGP 39393) at doses which prolong the activated partial thromboplastin time (APTT) to no more than 3–4 times the control level.In contrast, both unfractionated heparin and Fragmin only inhibit thrombosis when the APTT is excessively prolonged (i.e. to greater than 15 times the control value). It is concluded that CGP 39393 is an effective antithrombotic in arterial thrombosis at lower levels of anticoagulation than either heparin or Fragmin.
Effects of Different Hirudins and Combinations of Low Doses of Hirudin, Heparin and Acetylsalicylic Acid in a Rat Microcirculatory Thrombosis Model
Authors: Krupinski K. , Breddin H.K.
Published: April 2009

Two recombinant hirudins (r-hirudin), natural hirudin and hirudin in combination with heparin or acetylsalicylic acid (ASA) have been studied in a thrombosis model in which rat mesenteric venules of a diameter of 20–30 μm were injured by well-defined argon laser lesions. In the animal model all hirudins showed significant and dose-dependent antithrombotic effects in doses between 0.05 and 0.1 mg/kg after single intravenous and subcutaneous injections. The antithrombotic effect of single (0.2 mg/kg i.v. or 0.1 mg/kg s.c.) injections lasted longer than 4 h (i.v.) or 6 h (s.c). Hirudin at a dose of 0.1 mg/kg and heparin at doses of 0.05 mg/kg showed a significant antithrombotic effect 2 h after subcutaneous injection. When heparin and hirudin were injected together at this dosage, the effect of the combination was in the same range as that of unfractionated heparin or hirudin alone. An additive antithrombotic effect was observed if a low dose of r-hirudin (0.1 mg/kg) was combined with a moderate dose of ASA (10 mg/kg).
Effect of Recombinant Hirudin on Cytosolic Ca2+ Concentrations using Different Platelet Stimulators
Authors: Kirchmaier C.M. , Bellinger O.K. , Schulz B. , Breddin H.K.
Published: April 2009

Cytosolic Ca2+ concentration, [Ca2+]i, and aggregation behavior of hirudinized platelet suspensions were examined under stimulation with thrombin, adinosine diphosphate, platelet-activating factor, collagen and the calcium ionophore, ionomycin. A rise in [Ca2+]i, a key step in platelet activation, was only inhibited by recombinant hirudin r-hirudin using the agonist thrombin. All other stimulators including ionomycin were not affected by r-hirudin. If hirudinized blood was gel-filtered before platelet stimulation, thrombin induced the same response as citrated platelet suspensions. Aggregation studies and calcium influx using various stimulators showed good correlation: only thrombin-induced aggregation was completely inhibited by r-hirudin. These results support the hypothesis that hirudin inhibits platelet function by thrombin binding and not by direct interaction with platelets.
Pharmacokinetics, Effect on Clotting Tests and Assessment of the Immunogenic Potential of Hirudin after a Single Subcutaneous or Intravenous Bolus Administration in Man
Authors: Bichler J. , Siebeck M. , Fichtl B. , Fritz H.
Published: April 2009

The pharmacokinetics and the anticoagulant effects of hirudin were investigated in 12 healthy volunteers after single subcutaneous or intravenous bolus administrations. Hirudin concentrations in citrated plasma and urine were determined with a radioimmunobioassay, which detects the inhibitor by its thrombin-binding capactiy. Plasma profiles could be adequately described by the equation for an open two-compartment model (intravenous route) and by the Bateman equation (subcutaneous route), respectively. Within 24 h half of the administered hirudin dose was recovered in the urine in biologically active form. The prolongation of clotting times (activated partial thromboplastin and thrombin time) was dependent on the hirudin plasma concentration. All test subjects tolerated the hirudin injection without visible or measurable side effects. No hirudin-specific antibodies were found after single parenteral administrations.
Hirudin in Disseminated Intravascular Coagulation
Authors: Nowak G. , Markwardt F.
Published: April 2009

Various reactions of disseminated intravascular coagulation (DIC) were experimentally induced by infusion of thrombokinase in rats, by administration of endotoxin in rabbits and pigs and by infusion of adrenaline and thrombin in dogs. Low plasma concentrations of recombinant hirudin (r-hirudin; 20–200 ng/ml) were sufficient for the inhibition of the different triggering mechanisms. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy of this specific tight-binding thrombin inhibitor.
Hirudin for Diagnostic Purposes
Authors: Stocker K.
Published: April 2009

Hirudin serves as a versatile tool for the control of thrombin activity in hemostaseology. It may be added in excess to blood, plasma or test mixtures to prevent catalytic and nonenzymatic effects of thrombin. It may be used to quench thrombin activity upon extensive or limited action. Unlike heparin-antithrombin III complex which exclusively inhibits α-and γ-thrombin, hirudin also inhibits meizothrombin, a precursor of α-thrombin. Thus, hirudin may be used for the detection of meizothrombin as well as for the prevention of its action in plasma samples. In conjunction with chromogenic substrates, hirudin may serve to discriminate between actions mediated by thrombin, its precursors, cofactors and effectors and actions of other enzyme systems. The principle of this hirudin application is exemplified for factor-V- and factor-VIII-dependent anticoagulant activity of protein C.
Recombinant Hirudin – Initial Observations in Reconstructive Microsurgery
Authors: Narayanan K. , Walenga J.M. , Liang M.D. , Fareed J.
Published: April 2009

Recent advances in microvascular techniques have significantly enhanced the success rate of these procedures. Microvascular free tissue transfers at present have a 85–90% success rate. This, however, still leaves a 10–15% failure rate due primarily to thrombosis of the blood vessels at the anastomotic site. Previous work in the field has shown that anticoagulants such as heparin can reduce the rate of thrombosis. However, heparin can also cause on-site bleeding. Hirudin, a potent thrombin inhibitor of 65 amino acids, is available through recombinant technology. Our current experimental work utilized 75 male Sprague-Dawley rats for assessing the efficacy of hirudinized saline versus saline alone and heparinized saline as an irrigant in a microsurgery model of anastomosis. After exposing the right femoral artery in a standard fashion, the artery was clamped and a standard transverse division of the vessel was made. The vessel ends were irrigated with one of the three solutions then anastomosed. Vessels were observed for patency over a 3-day period. Initial results showed comparable performance of hirudinized saline and heparized saline, both of which were better than saline, for preventing thrombosis. From these initial data, and the reduced bleeding risk associated with hirudin, this agent appears to be a useful anticoagulant in microsurgery.
PECAM-1 Is Necessary for Flow-Induced Vascular Remodeling
Authors: Chen Zhongming , Tzima Ellie
Published: April 2009

Objective—

Vascular remodeling is a physiological process that occurs in response to long-term changes in hemodynamic conditions, but may also contribute to the pathophysiology of intima-media thickening (IMT) and vascular disease. Shear stress detection by the endothelium is thought to be an important determinant of vascular remodeling. Previous work showed that platelet endothelial cell adhesion molecule-1 (PECAM-1) is a component of a mechanosensory complex that mediates endothelial cell (EC) responses to shear stress.

Methods and Results—

We tested the hypothesis that PECAM-1 contributes to vascular remodeling by analyzing the response to partial carotid artery ligation in PECAM-1 knockout mice and wild-type littermates. PECAM-1 deficiency resulted in impaired vascular remodeling and significantly reduced IMT in areas of low flow. Inward remodeling was associated with PECAM-1-dependent NFκB activation, surface adhesion molecule expression, and leukocyte infiltration as well as Akt activation and vascular cell proliferation.

Conclusions—

PECAM-1 plays a crucial role in the activation of the NFκB and Akt pathways and inflammatory cell accumulation during vascular remodeling and IMT. Elucidation of some of the signals that drive vascular remodeling represent pharmacologically tractable targets for the treatment of restenosis after balloon angioplasty or stent placement.
Potential Animal Models of Lacunar Stroke
Authors: Bailey Emma L. , McCulloch James , Sudlow Cathie , Wardlaw Joanna M.
Published: April 2009

Background and Purpose—

Lacunar ischemic stroke accounts for 25% of all ischemic strokes, but the exact etiology is unknown. Numerous pathophysiologies have been proposed, including atheroma and endothelial dysfunction. Models of any of these pathological features would aid understanding of the etiology and help develop treatments for lacunar stroke. We therefore aimed to assess the relevance of all available potential animal models of lacunar stroke.

Methods—

We systematically reviewed the published literature for animal models that could represent lacunar stroke using validated search strategies. We included studies that could represent an aspect of lacunar stroke as well as those aiming to model conditions with potentially similar pathology and extracted data on species, induction method, and resulting brain and vessel lesions.

Results—

From 5670 papers, 41 studies (46 papers) met inclusion criteria representing over 10 different classes of stroke induction. Important data like infarct size and animal numbers were often missing. Many models’ infarcts were too large or affected the cortex. Emboli mostly caused cortical but not small subcortical lesions. Most models focused on creating ischemic lesions in brain tissue. Only one (spontaneous lesions in spontaneously hypertensive stroke-prone rats) also mimicked small vessel pathology. Here, the precursor to small vessel and brain damage was blood–brain barrier failure.

Conclusion—

Some animal models produce small subcortical infarcts, but few mimic the human small vessel pathology. Models of small vessel disease could help improve understanding of human lacunar disease, particularly to clarify factors associated with the small vessel morphological changes preceding brain damage. Much lacunar stroke may arise after blood–brain barrier disruption.
Effect of Antiplatelet Therapy for Endovascular Coiling in Aneurysmal Subarachnoid Hemorrhage
Authors: van den Bergh Walter M. , Kerr Richard S.C. , Algra Ale , Rinkel Gabriel J.E. , Molyneux Andrew J.
Published: April 2009

Background and Purpose—

Antiplatelets are frequently used during or after endovascular coiling of aneurysm in patients with subarachnoid hemorrhage (SAH). This strategy is based on uncontrolled case series including also patients with unruptured aneurysms or other lesions. We collected data on effectiveness of antiplatelets in patients with SAH.

Methods—

All 43 participating centers in the International Subarachnoid Aneurysm Trial (ISAT) were sent a questionnaire whether they never, sometimes, or always prescribed antiplatelets during or after coiling. Based on individual patient data, the relative risks (RRs) of coiling versus clipping were calculated separately for patients treated in hospitals with standard prescription during or after coiling versus patients treated in hospitals with no standard prescription of antiplatelets. We calculated ratios of RRs for standard versus not standard prescription of antiplatelets during coiling and for standard versus not standard prescription after coiling.

Results—

Nineteen centers responded, representing 1422 (66%) of the 2143 ISAT patients. Antiplatelets were standard prescribed during coiling in 2 responding centers (8% of coiled patients) and after coiling in 6 centers (24%). For poor outcome at 2 months of coiling versus clipping the RR was 0.82 (95% CI: 0.45 to 1.49) in hospitals with a policy of antiplatelet prescription during coiling versus 0.66 (95% CI: 0.55 to 0.78) in those without such policy (ratio of RR’s 1.24,
P
=0.56). The ratio of RRs for 1-year outcome was 1.01 (
P
=0.89) for antiplatelet use during coiling and 1.00 (
P
=0.77) for use after coiling.

Conclusion—

The results of this study do not support the assumption that antiplatelets during or after endovascular coiling improve outcome in patients with SAH.