bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetics of Heparin and Low Molecular Weight Heparins
Authors: Ambrosioni E. , Strocchi E.
Published: April 2009

The main kinetic features of heparin and low molecular weight heparins (LMWHs) are reviewed. The complexities related to the study of a nonhomogeneous drug containing fractions with different molecular weight and pharmacological effects are discussed along with the possible bias due to the prevalent use of indirect measures of drug concentration. Available data show that LMWHs have longer half-lives with respect to that of heparin and higher bioavailabilities after subcutaneous administration; experimental and clinical evidences suggest that the kinetics of heparin and LMWHs can only be described taking into account the presence of an endogenous pool from which these drugs are released with different speeds and after various types of manipulations.
Heparin, Monocytes, and Procoagulant Activity
Authors: Abbate R. , Gori A.M. , Modesti P.A. , Attanasio M. , Martini F. , Colella A. , Giusti B. , Cecioni I. , Neri Serneri G.G.
Published: April 2009

Monocytes are able to specifically bind heparin rapidly, reversibly, and saturably with 5.5 ± 2.6 × 106 binding sites per monocyte and a dissociation constant of 330 ± 221 nmol/l. Moreover, at least 40–50% of the cell-bound heparin can be internalized. Heparin binding by monocytes is affected by cell stimulation with an increase of the availability of binding sites after challenge with calcium ionophore, lipopolysaccharide, and interleukin 2. The interaction of heparin with monocytes reversibly decreases the expression of tissue factor on the cellular surface, so hampering the cellular procoagulant potential.
Heparin in Acute Myocardial Infarction
Authors: Tavazzi Luigi
Published: April 2009

Thrombolytic therapy has become an established procedure in patients with acute myocardial infarction (AMI). However, the aftercare of such patients is still uncertain. A meta-analysis of twenty trials of intravenous or subcutaneous heparin in AMI performed during the prethrombolysis period indicated a significant reduction in mortality, reinfarction, and stroke in treated patients. More recently, a study (SCATI) aimed at investigating the clinical effects of subcutaneous heparin (12,500 U two times daily) in the setting of thrombolytic therapeutical strategy in AMI, showed a lower in-hospital mortality, a trend towards lesser transient ischemic episodes in patients given streptokinase, and no difference in recurrent infarction rate. Ventricular thrombi were markedly reduced by heparin. Anti-platelet drugs were not permitted in the SCATI. In both GISSI 2 (Gruppo Italiano per lo Studio della Streptochinasi nell’Infarto Miocardico) and International tissue plasminogen activator/streptokinase trials, aspirin was a recommended treatment, and subcutaneous heparin was randomized. No difference in mortality was noted in patients given heparin. In GISSI 2 recurrent ischemic episodes were also similar in treated and control groups, whereas embolic events were reduced by heparin. Major bleedings were rare in all trials. In conclusion, subcutaneous heparin is beneficial in AMI; however, the association with aspirin does not add consistent benefits. Heparin is effective in preventing thrombus formation and embolic complications – such effects are not shared by aspirin.
Approaches to the Synthesis of Heparin
Authors: Lindahl Ulf
Published: April 2009

The biosynthesis of heparin is initiated by formation of [GlcA-GlcNAc]n polysaccharide chains linked to the core protein of a proteoglycan structure. The polymer is transformed into the mature polysaccharide by a series of modification reactions which involve N-deacetylation and N-sulfation of GlcN Ac units, C5 epimerization of GlcA to IdoA residues, and O-sulfation at different positions. Incomplete modification, controlled in part by the substrate specificities of the corresponding enzymes, provides the complex saccharide sequences that are typical for heparin and, in particular, for heparan sulfate. One such structure is the antithrombin-binding region which is comprised by a specific pentasaccharide sequence with a 3-O-sulfated GlcN marker group. Aspects of regulation of polymer modification are discussed.
Heparin by Alternative Routes of Administration
Authors: Andriuoli G. , Bossi M. , Caramazza I. , Zoppetti G.
Published: April 2009

In a study investigating the bioavailability of heparin administered by different routes, we also compared its bioavailability in the dog after oral administration of two enteric-coated formulations, one containing a heparin preparation (ITF-300) and the other sodium heparin. After administration of the formulation containing sodium heparin, there was no heparin in plasma, but when the formulation containing ITF-300 was given, plasma heparin levels were detectable.
Heparin-Endothelial Cell Interactions
Authors: D’Amore Patricia A.
Published: April 2009

Recent observations have revealed that heparin influences endothelial cell proliferation in a number of ways unrelated to its anticoagulant properties. A majority of the nonanticoagulant actions of heparin on the endothelium appear to be related to heparin interactions with the fibroblast growth factors (FGFs). Members of this family of potent endothelial mitogens and angiogenic factors have been shown to bind with high affinity to immobilized heparin. It was this characteristic of the FGFs that initially allowed their purification to homogeneity. Subsequently, it has been shown that a unique characteristic of the FGFs is their lack of a signal sequence which results in their not being secreted by conventional means. However, both in vitro and in vivo studies have revealed that a significant proportion of the cell-synthesized FGF is found outside the cells, localized in the extracellular matrix bound to heparin-like glycosaminoglycans. These findings have led to the suggestion that matrix-associated FGF represents an extracellular reservoir that may be released by the action of heparan sulfate degrading enzymes introduced during inflammation or tumor metastasis or by heparin that is released from mast cells. We have recently shown that intravenous infusion of heparin into rabbits results in an increase in plasma levels of an FGF-like molecule. Other studies, in which investigators have infused FGF into normal animals, have revealed no effect of the circulating FGF on cells of a normal adult vasculature. However, release of FGF into the circulation, where there has been ‘compromise’ in the vascular system, indicated a dramatic influence on the rate of proliferation of vascular cells at the injured sites. In addition, heparin has been shown by a number of investigators to potentiate the action of acidic FGF by protecting the peptide from denaturation. We have recently shown that the potentiation can be obtained by heparin fragments and that it is not dependent on the anticoagulant activity of heparin, but is correlated with the degree of sulfation. The ability of heparin to protect FGF may have physiologic impact in microenvironments where FGF is released in the presence of locally high heparin concentrations, thereby prolonging its biological half-life. Taken together, these observations indicate a role for heparin, distinct from its anticoagulant activity, in regulating the growth of vascular endothelial cells, both by modulating the availability and stability of potent growth-regulating agents.
Effectiveness and Safety of the Low-Molecular-Weight Heparin CY 216 in the Prevention of Fatal Pulmonary Embolism and Thromboembolic Death in General Surgery
Authors: Pezzuoli G. , Neri Serneri G.G. , Settembrini P.G. , Coggi G. , Olivari N. , Negri G. , Codemo R. , Galli G. , Roveri S.
Published: April 2009

Deep venous thrombosis is very frequent after general surgery, and its major complication, pulmonary embolism, is today the most frequent cause of postoperative death. The reduction of this cause of mortality is mainly based on its prevention rather than its therapy. This purpose was achieved by using physical and pharmacological means. During
Anticardiolipin Antibodies in Patients with Venous Thrombosis
Authors: Kapiotis S. , Speiser W. , Pabinger-Fasching I. , Kyrle P.A. , Lechner K.
Published: April 2009

The levels of anticardiolipin antibodies (ACA) and lupus anticoagulant (LAC) were measured in 266 consecutive unselected patients with a history of venous thrombosis. 19 (7.1 %) had elevated levels of IgG- or IgM-ACA. The prevalence of LAC was 1 of 266 (0.4%) in the whole patient group and 1 of 19 (5.3%) in the ACA-positive group. Patients with elevated ACA levels did not differ from those with normal ACA with regard to age at the first thrombosis, risk of recurrence, presence of arterial thrombosis, and other clinical features. 8 out of 19 (42.1%) patients with elevated ACA levels also had elevated levels of antinuclear antibodies, but only 1 fulfilled the criteria of systemic lupus erythematosus. These data indicate that in some patients with elevated ACA, autoimmune processes may be present. The clinical significance of elevated ACA levels is uncertain.
Separation of Lupus Anticoagulant from Anticardiolipin Antibodies by Ion-Exchange and Gel Filtration Chromatography
Authors: Chamley L.W. , Pattison N.S. , McKay E.J.
Published: April 2009

Separation of lupus anticoagulant from anticardiolipin antibodies in the serum of a patient containing both antibodies is described. A simple two-step procedure utilizing diethylaminoethylcellulose ion-exchange chromatography followed by Sepharose CL-4B gel filtration chromatography allowed the separation of IgM from IgG isotype. Lupus anticoagulant was found to be exclusively IgM, whilst anticardiolipin antibodies were IgG. This is apparently the first report that anticardiolipin antibodies and lupus anticoagulant can be different isotypes and adds to the increasing evidence that they are separate entities.
Plasma Homocysteine in Venous Thromboembolism
Authors: Brattström Lars , Tengborn Lilian , Lagerstedt Claes , Israelsson Bo , Hultberg Björn
Published: April 2009

Severe hyperhomocysteinemia due to inborn errors of methionine metabolism results in precocious development of arteriosclerosis and predisposition to venous and arterial thromboembolism. Although the findings of several studies have indicated that mild hyperhomocysteinemia is common in occlusive arterial disease, no similar studies have been made on venous thromboembolism. In this study of subjects under 50 years of age, we found no significant differences in the plasma homocysteine concentrations, either in the fasting state or after methionine loading, between 42 patients with venous thromboembolism and 42 healthy controls. Nonetheless, male patients manifested a tendency toward higher homocysteine concentrations than male controls; 6 patients (14%) versus 2 controls (5%) responded abnormally to methionine loading which might indicate heterozygosity for cystathionine synthase deficiency. Thus, further studies on plasma homocysteine in venous thromboembolism are warranted.
Intra- and Postoperative Fibrinolysis in Patients Undergoing Cardiopulmonary Bypass Surgery
Authors: Páramo J.A. , Rifón J. , Llorens R. , Casares J. , Paloma M.J. , Rocha E.
Published: April 2009

The influence of cardiopulmonary bypass (CPB) on fibrinolytic activity was assessed in 100 patients with valvular heart disease or atrial septal defects. Euglobulin fibrinolytic activity (EFA), tissue type plasminogen activator (t-PA) activity, plasminogen activator inhibitor 1 (PAI-1) activity, plasminogen, α2-antiplasmin (α2-AP), fibrinogen degradation products (FDP), and D-dimer were measured pre-, intra-, and postoperatively. There were significant increases in EFA and t-PA activity (p < 0.002), and decreases in plasminogen and α2-AP (p < 0.0001) intraoperatively with respect to baseline values. t-PA activity decreased significantly after surgery (p < 0.002), whereas PAI-1 activity showed a marked increase shortly after operation and on postoperative day 1 (p < 0.0001). FDP and D-dimer levels were significantly increased both intra- and postoperatively, the latter showing higher values (p < 0.01 and p < 0.0001, respectively). This study shows that there is an activation of the fibrinolytic system, as a result of the increased activation of plasminogen and decreased levels of plasmin inhibitors, during CPB surgery followed by a postoperative fibrinolytic shutdown.
Antithrombin III Activity Is Not Related to Plasma Homocysteine Concentrations
Authors: Bienvenu T. , Chadefaux B. , Ankri A. , Leblond V. , Coude M. , Salehian B. , Binet J.L. , Kamoun P.
Published: April 2009

Arterial and venous thromboembolic events represent frequent and life-threatening complications in homocystinuric patients and are responsible for their early deaths. Reduced levels of antithrombin III activity in homocystinuric patients have recently been reported. So, high plasma L-homocysteine concentration could play a role in the low antithrombin III activity level. In the present study, we have studied the relationship between total plasma homocysteine and inhibitors of blood coagulation levels in 16 patients with malignancies who received bone marrow grafts. There were no correlations between homocysteine values and inhibitors of blood coagulation levels. So, while the defect in amino acid transsulfuration that is responsible for homocystinuria can directly affect the synthesis or activity of some clotting factors, homocysteine concentration is not responsible for this effect.
Kinetic Aspects of Release of Fibrinopeptides AP and AY by Human Alpha-Thrombin
Authors: De Cristofaro Raimondo , Castagnola Massimo
Published: April 2009

The time-dependent release by human α-thrombin of the physiological variants of fibrinopeptide A, i.e. fibrinopeptide A-3-phosphate (FPAP) and des-Ala-fibrinopeptide A (FPAY), has been measured in order to study the kinetic pathway for their hydrolysis. The best-fit kinetic model for the release of FPAP and FPAY is consistent with a simple pseudo first-order reaction, as observed with FPA. These findings indicate that FPAP and FPAY are also released from intact fibrinogen before release of FPB. The values of the specificity constants, i.e. kat/Km, for the enzymatic reaction between thrombin and the various α-chains showed that APα- and AYα-chain are hydrolyzed with a 0.2-and 0.4-fold higher specificity constant respectively than that of Aα-chain. Such minor differences observed with the various chains suggest that the 1–3 NH2-terminal residues of the chain do not significantly contribute to the catalytic efficiency of thrombin toward the α-chains of fibrinogen.
Coagulation/Fibrinolysis Balance and Lung Cancer
Authors: van Wersch J.W.J. , Tjwa M.K.T.
Published: April 2009

Forty-eight patients with freshly diagnosed carcinoma of the lung (40 males, 8 females) were evaluated for a coagulation profile including activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, F VIII R:Ag, fibrin monomers (FM), thrombin-antithrombin-III complex (TAT-III), D-dimers and the platelet count. Thirty-eight patients had a normal aPTT and 37 patients a normal PT. None of the patients had clinical or laboratory indications of serious hemorrhage or thrombosis. On the other hand, high percentages of increased values were found for fibrinogen and F VIII R:Ag, which can be seen as prethrombotic factors. The very high percentages of elevated results for the FM, TAT-III and .D-dimer are strongly indicative for low-grade coagulation activation with reactive fibrinolysis. Nevertheless, most lung cancer patients are able to maintain a normal or near normal hemostatic function. The results shown here are indicative of a coagulation and fibrinolysis equilibrium at an enhanced level and demonstrate why an unbalance between the two systems can result in thrombotic complications in (lung) cancer patients as earlier reported.
Measurement of von Willebrand Factor Antigen in Plasma and Platelets with an Enzyme-Linked Immunosorbent Assay Based on Two Murine Monoclonal Antibodies
Authors: Tornai I. , Declerck P.J. , Smets L. , Arnout J. , Deckmyn H. , Caekebeke-Peerlinck K.M.J. , Vermylen J.
Published: April 2009

Two murine monoclonal antibodies, raised against von Willebrand factor (vWF), were used to construct an enzyme-linked immunosorbent assay (ELISA), for quantitation of vWF antigen (vWFAg) in human plasma and platelets. This assay had a lower limit of sensitivity of 0.0001 IU/ml in buffer, and thus is one to two orders of magnitude more sensitive than other ELISA assays which have been reported. The intraassay, interassay and interdilution coefficients of variation were 4.1, 10.4 and 9.9%, respectively. In normal plasma (n = 20), the vWFAg level was 0.83 (range: 0.42–1.25) IU/ml. In normal washed platelets (n = 10), 0.35 (0.25–0.49) IU/109 platelets was found. In plasma obtained from various patient groups the following vWFAg levels (geometric mean and range) were observed: von Willebrand’s disease (n = 19): 0.18 (0.02–0.77) IU/ml; patients with liver cirrhosis (n = 20): 3.73 (1.68–9.20) IU/ml; patients with pregnancy-induced hypertension (n= 20): 4.14 (2.28–7.44) IU/ml and patients with malignant disease (n = 10), 2.54 (1.51–5.60) IU/ml. A linear correlation was found between vWFAg levels measured with a polyclonal antibody based Laurell electroimmunoassay (r = 0.92, n = 58) or with a polyclonal antibody based ELISA (r = 0.94, n = 64). The present assay is based on stable and reproducible reagents and allows the specific measurement of vWFAg in plasma and in platelets. This assay may constitute a useful tool for the further investigation of clinical conditions associated with changes in vWFAg levels. In addition, its high sensitivity may facilitate a more detailed study of platelet vWFAg in normal and in pathological conditions.
Effect of Fibrinogen Fragments D and E on the Adhesive Properties of Human Granulocytes to Venous Endothelial Cells
Authors: Fischer E.G. , Vogel P. , Ziegler A.S. , Kirkpatrick C.J.
Published: April 2009

In hyperfibrinolytic conditions, e.g. in disseminated intravascular coagulation or the adult respiratory distress syndrome, high levels of fibrinogen degradation products (FDPs) D and E are found in human plasma. This study investigates the influence of these fragments on cell attachment of human granulocytes in vitro. While leaving unaffected the adhesion of human umbilical vein endothelial cells (HUVEC) on gelatine-coated glass, both FDP fragments at 50 μg/ml inhibited granulocyte attachment to glass as well as to HUVEC monolayers. At the same concentration, the fragments diminished the superoxide release of stimulated granulocytes. These results suggest a modulatory role of pathologically elevated FDPs on the granulocyte function cascade.
Effective Prophylaxis of Thrombosis by Antithrombin III Concentrate in a Pregnant Woman with Congenital Antithrombin III Deficiency: Relations between Plasma Antithrombin III Activity and the Plasma Levels of Hemostatic Molecular Markers
Authors: Inomoto T. , Takamoto M. , Tamura R. , Maegawa M. , Kamada M. , Takayanagi M.
Published: April 2009

The value of antithrombin III (AT III) concentrate and a standard criterion for its use were examined in a pregnant woman with congenital AT III deficiency by continuous monitoring of plasma AT III activity and the plasma levels of hemostatic molecular markers. The rates of improvement of various markers after AT III administration (frequency of improvement/frequency of administration) were as follows: fibrinopeptide A (FPA) 82%, .D-dimer 70%, fibrinopeptide B β15-42 73%, β-thromboglobulin 60%, and platelet factor 4 50%. There was no significant correlation between the plasma AT III activity and all plasma FPA values, but FPA values of over 3.9 ng/ml showed a significant negative correlation with AT III activity: AT III activity (%) = -6.59 × FPA (ng/ml) + 125, r = -0.851, p < 0.02. We therefore recommend continuous monitoring of the plasma FPA level and administration of AT III concentrate when the FPA level is elevated. According to the regression line shown above, plasma AT III activity should be raised to 100% to keep the FPA level below 6.0 ng/ml with 95% confidence limit.
An Open Trial of Enoxaparin in the Treatment of Deep Vein Thrombosis of the Leg
Authors: Janvier G. , Freyburger G. , Winnock S. , Dugrais G. , Boisseau M. , Boissiéras P.
Published: April 2009

An open and prospective phase II trial assessing the action of a fixed dose of a low molecular weight heparin (enoxaparin), determined by the patient’s weight, in the treatment of established deep vein thrombosis of the leg is hereby described. A series of 51 patients received a regimen of 1 mg•kg-1 or 105 anti-Xa IU•kg-1 s.c. every 12 h for a period of 12 days. Thromboses were categorized as postsurgical (28 cases) or medical (23 cases). There was a significant improvement in clinical signs (pain and edema) and phlebographic indices (Marder and Arnesen scores). The extent of vascular clearing was a 30% reduction in phlebographic scores between day 0 and day 12. Only 5 patients had an absence of improvement. Two of the 51 patients stopped the drug in the first week of treatment because of bleeding. There were no occurrences of thrombocytopenia, and the agent was well tolerated.
Hydrogen Peroxide as Trigger of Platelet Aggregation
Authors: Praticò D. , Iuliano L. , Ghiselli A. , Alessandri C. , Violi F.
Published: April 2009

In order to verify if H2O2 affects platelet function, platelet-rich plasma and human washed platelets were incubated with subthreshold concentrations (STC) of collagen or arachidonic acid or ADP and/or with 75–150 μM H2O2. While H2O2 alone did not affect platelet aggregation, it amplified platelet aggregation response in samples stimulated with STC of arachidonic acid and collagen but not in samples stimulated with STC of ADP. When platelets were preventively treated with aspirin, a cyclooxygenase inhibitor, the platelet activation by H2O2 was not observed. Thromboxane A2 (TxA2) was not produced by human washed platelets stimulated with STC of arachidonic acid, collagen or by H2O2 alone. On the contrary, when STC of agonists were tested on platelets supplemented with H2O2 an evident TxA2 production was seen. This effect was prevented by aspirin pretreatment or by the addition of catalase, an enzyme which destroys H2O2. This study suggests that H2O2 triggers the activation of platelets exposed to STC of collagen and arachidonic acid, via the cyclooxygenase pathway.
Diet-Induced Changes in Glucose and Triglycerides Are Associated with Changes in Plasminogen Activator Inhibitor Levels
Authors: Sundell Birgitta , Dahlèn Gösta H. , Rånby Mats
Published: April 2009

Twenty-four healthy female subjects volunteered to participate in an experiment in which they maintained a high-fat/low-carbohydrate (CHO) diet followed by a low-fat/high-CHO diet or vice versa in a cross-over study design. In bivariate correlation analysis, only glucose (r = 0.52, p < 0.05) and triglycerides (r = 0.53, p < 0.05) correlated with changes in plasminogen activator inhibitor type 1 (PAI-1) activity. A second-degree polynomial response surface model suggested that transition from a high-fat/low-CHO diet to a low-fat/high-CHO diet is associated with reduced levels of PAI-1 provided that glucose and triglyceride levels are not elevated by more than 1.2 and 0.5 mmol/l, respectively.
Intranasal DDAVP: Biological and Clinical Evaluation in Mild Factor VIII Deficiency
Authors: Chistolini A. , Dragoni F. , Ferrari A. , La Verde G. , Arcieri R. , Ege Mohamud Ali , Mazzucconi M.G.
Published: April 2009

We report a study undertaken to test the biological effect of intranasal 1 -deamino-8-.D-arginine vasopressin (DDAVP) and its efficacy in the treatment of bleedings in patients with mild factor VIII deficiency. The biological study was carried out in 20 patients: an increase of factor VIII:C and von Willebrand factor antigen levels was observed after inhalation of DDAVP at average post/pre inhalation ratios of 2.80 and 1.72, respectively. No relevant alterations of fibrinolysis were noted. In fact, we only observed a simultaneous increase of tissue plasminogen activator and plasminogen activator inhibitor, without modification of D-dimer. In 10 cases intranasal DDAVP has been used in the prevention or in the treatment of bleeding complications: no bleedings were observed.
The Beneficial Effect of Lys-Plasminogen upon the Thrombolytic Efficacy of Urokinase in a Dog Model of Peripheral Arterial Thrombosis
Authors: Badylak Stephen F. , Voytik Sherry L. , Henkin Jack , Burke Sandra , Sasahara Arthur A. , Simmons Abby
Published: April 2009

The efficacy of thrombolytic therapy may be limited by local availability of plasminogen near a poorly perfused thrombus. The purpose of this study was to determine if the local (i.e., clot site) administration of 0.5 mg glu-plasminogen (glu-plg) or 0.5 mg lys-plasminogen (lys-plg) could safely increase the thrombolytic efficacy of a 30-min intraarterial injection of 3,500 U kg-1 of two-chain urokinase plasminogen activator (UK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by a distally placed flowmeter and by direct visual examination. Forty-two dogs (mean weight 10.1 ± 1.9 kg) were randomly sorted into six groups of 7 each. The dogs in each group were given either saline plus saline (group 1), saline plus UK (group 2), glu-plg plus saline (group 3), glu-plg plus UK (group 4), lys-plg plus saline (group 5), or lys-plg plus UK (group 6) by selective arterial catheterization 60 min after formation of an occlusive thrombus. Ninety minutes following drug administration, all groups which received UK (groups 2, 4, and 6) showed greater lysis (p < 0.05) than the groups which received only saline or either glu- or lys-plg plus saline. Group 6, which received lys-plg plus UK, showed significantly greater lysis (34 ± 4%) than both group 2 (23 ± 2%), which received saline plus UK, and group 4 (19 ± 3 %), which received glu-plg plus UK (p < 0.05). All dogs (7/7) in group 6 had reflow at 90 min whereas only 3/7 dogs had reflow in both groups 2 and 4. None of the dogs in groups 1, 3, or 5, which received only saline or either form of plasminogen, had reflow at 90 min. There were no changes in the prothrombin time, activated partial thromboplastin time, thrombin time, hematocrit, or platelet count in any of the six groups. We conclude that lys-plg can safely increase the thrombolytic efficacy of UK in a dog model of arterial thrombosis.
Diagnostic Value of D-Dimer for Deep Venous Thrombosis in Outpatients
Authors: Kroneman Herman , Van Bergen Paul F.M.M. , Knot Eduard A.R. , Jonker Jan J.C. , de Maat Moniek P.M.
Published: April 2009

We have studied the diagnostic value for deep venous thrombosis (DVT) of an enzyme immunoassay (EIA) for the detection of D-dimer in plasma of 239 consecutive outpatients suspected of having DVT by their general practitioner. DVT was confirmed by impedance plethysmography in 60 patients. Using the 95th percentile range of 42 healthy volunteers, the sensitivity for the detection of DVT was 92 %, with a specificity of 21 %. In our population with a prevalence of 25%, the D-dimer EIA showed a negative predictive value of 88 % and a positive predictive value of 28%. We conclude that this D-dimer ELISA has limited value, either to confirm or to exclude DVT in outpatients.
Thromboembolic Risk in Children with Nephrotic Syndrome
Authors: De Mattia Domenico , Penza Rosa , Giordano Paola , Di Bitonto Giovanna , Altomare Maria , Del Vecchio Giovanni C. , Schettini Francesco
Published: April 2009

The in vivo activation of the hemostatic system was evaluated in 14 children (4–13 years old) with nephrotic syndrome at different stages of the disease. The blood platelet count, β-thromboglobulin (β-TG), platelet factor 4 (PF4), fibrinogen, the coagulation inhibitors antithrombin III and protein C (ATIII:Ag and PC:Ag), and D-dimers were determined. Platelet number was significantly higher at the onset of the disease than in the next stages (p < 0.05). β-TG, PF4 and fibrinogen were significantly increased as compared with controls at the onset (p < 0.001) and decreased progressively during the course of the disease without reaching the control values. Blood coagulation inhibitors behaved differently; PC was higher in patients than in controls at all stages (p < 0.05) whereas ATIII values were significantly decreased at the onset (p < 0.05), but increased during the course the disease (p < 0.01). No changes were observed in the D-dimer plasma levels. These data suggest that the thrombotic risk in nephrotic syndrome is particularly evident at the onset of the disease, and appears to be due mainly to changes in platelet number and function, and to increased fibrinogen levels rather than to alterations of plasma anticoagulant factors.
Effects of Epsilon Aminocaproic Acid on Primary Hemostasis
Authors: Zeigler Z.R.
Published: April 2009

These studies examined the effect of an oral dose of ε-aminocaproic acid (EACA) on primary hemostasis. Bleeding time tests (with and without the use of a blood pressure cuff) were measured before and 2 h following EACA in 56 patients with mild bleeding disorders and/or thrombocytopenia. Preliminary studies evaluated the reproducibility of these tests in 13 patients who had bleeding times (cuff) ranging from 8.0 to > 20 min. Their replicate bleeding time values with cuff agreed within 2.5 min and those without cuff within 3 min. Therefore, the 56 study patients were considered to have had no change in their bleeding times after EACA, if their bleeding time with cuff was ± 2.5 min and/or their nonoccluded value was ± 3 min of their baseline values, respectively. An isolated increase in bleeding times was observed in 6 of 56 (11 %) patients. All 6 had myelodysplasia associated with long bleeding times; their nonoccluded values increased by 5–14 min. Of the 56 study patients, 54% showed a decrease in their bleeding times following EACA. The changes were evident with venostasis in 18 of 30 (60 %) and without venostasis in 12 (40%) patients. These studies suggest that EACA may improve primary hemostasis in some patients with prolonged bleeding times.
Effect of Mental Stress on Platelet Aggregation: Possible Link to Catecholamine Levels
Authors: Pietraszek M.H. , Takada Y. , Takada A.
Published: April 2009

The present study was aimed at testing the hypothesis about the relation between mental stress and platelet function. Plasma samples were obtained from 100 students immediately before, and 30 min and 7 h after undergoing a bleeding time test, which was used as mental stressor. Platelet responses to serotonin, ADP and collagen, as well as plasma catecholamine levels were assayed. Plasma serotonin levels had a high negative correlation with the bleeding time. Stress resulted in an increase in platelet responses to serotonin, ADP and to the elevation of catecholamine levels. Furthermore, a significant negative correlation between the platelet aggregation induced by ADP and collagen and the bleeding time was demonstrated. It is concluded that mental stress had a significant effect on platelet aggregation. This study provides evidence of increased platelet aggregability in association with elevated plasma catecholamine levels.
Past, Present and Future of Hirudin
Authors: Markwardt Fritz
Published: April 2009

The naturally occurring anticoagulant from medicinal leeches, hirudin, which we isolated and biochemically analyzed 30 years ago as a miniprotein with specific anti-thrombin activity, has afterwards been employed for scientific and diagnostic purposes in hematology. Pure hirudin proved to be an antithrombotic agent of high quality that displays an antithrombotic action dependent upon its blood level. After intravenous injection, it is distributed in the extracellular space and is almost completely eliminated through the kidneys by glomerular filtration in a biologically active form. The efficacy of hirudin in preventing venous and arterial thrombosis and disseminated intravascular coagulation was demonstrated in various animal models. Clinical pharmacological studies corroborated the specific pharmacodynamic and pharmacokinetic properties of hirudin found in animal experiments. Genetic engineering led to the availability of sufficient quantities of recombinant hirudin (r-hirudin) for clinical purposes. Pharmacologic profiling of r-hirudin showed that both its pharmacokinetic and pharmacodynamic characteristics are very similar to those of native hirudin. Clinical pharmacological studies with r-hirudin revealed that, at single therapeutically relevant doses, r-hirudin is a well-tolerated and potent anticoagulant without any detectable side effects and allergic reactions. Further preclinical studies of r-hirudin should concentrate on identifying possible indications for use, on the development of r-hirudin preparations and derivatives, and on the development of antidotes for hirudin.
Thrombin Inhibition by Hirudin: How Hirudin Inhibits Thrombin
Authors: Fenton II J.W. , Villanueva G.B. , Ofosu F.A. , Maraganore J.M.
Published: April 2009

In addition to its classical active-site regions (catalytic site and adjacent regions), α-thrombin has a unique anion-binding exosite, which is functionally independent of the catalytic site and is involved in fibrin(ogen) recognition. This exosite also accounts for adhesion to negatively charged surfaces (e.g., glass), binding to cell surfaces, and interactions with the anionic tail of hirudin. Hirudin (as an apolar, tridisulfide-linked core structure followed by its anionic tail) interacts with α-thrombin by apolar (e.g., catalytic-site and adjacent regions of thrombin), as well as by ionic binding (e.g., anion-binding exosite). Circular dichroism measurements reveal a sigmoidal nonadditivity for the hirudin tail fragments, which block fibrinogen-clotting activity without interfering with tripeptide chromogenic substrate activities. Such fragments, however, inhibit factor V activation to much lesser extents than hirudin, where factor V activation is the key step in regulating thrombin generation by hirudin or heparin/antithrombin III. Hirudin-derived antithrombotics may thus have differential modes of action in hemostasis and wound healing processes.
Structure-Function and Refolding Studies of the Thrombin-Specific Inhibitor Hirudin
Authors: Johnson Paul H. , Sze Ping , Winant Richard C. , Hudson Debra , Underhill Peter , Lazar Jerome B. , Olsen Cris , Almquist Ron
Published: April 2009

We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human α-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration ( > 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudimthrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42–65 and 51–65 [but not (Ala22)-6–28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 μM, respectively, at a thrombin concentration of 1μM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.
Antithrombotic Effects of Recombinant Hirudin in Different Animal Models
Authors: Just Melitta , Tripier Dominique , Seiffge Dirk
Published: April 2009

The effect of recombinant hirudin (r-hirudin; HBW 023) was studied in four different models of thrombosis as well as on bleeding time. Arteriolar thrombus formation was induced in rats either by argon laser injury or by photochemical reaction. r-Hirudin, given by intravenous infusion, significantly inhibited arteriolar thrombus formation and arterial bleeding time at a dose of 40 μg/kg·min. In rabbit jugular veins and carotid arteries, occluding thrombi were produced by stenosis and endothelial damage. r-Hirudin reduced the incidence of thrombosis dose dependently (ED50 0.7 and 1.0 mg/kg i.v. in arteries and veins, respectively). Caval vein thrombosis was initiated in rats by insertion of a stainless steel coil. This thrombosis was dose dependently reduced by the injection of r-hirudin (ED50 0.16 mg/kg i.v.). The duration of the antithrombotic effect of 0.25 mg/kg of r-hirudin in caval veins was about 60 min, whereas the arterial bleeding time was significantly prolonged in these rats for only 20 min. The plasma level of r-hirudin had dropped to 0.12 μg/ml 60 min after injection. These results suggest that the duration of the effect on bleeding time may be shorter than the antithrombotic action of r-hirudin.
Effect of Recombinant Hirudin (LU 52369) on Reocclusion Rates after Thrombolysis in Rabbits
Authors: Rübsamen K. , Eschenfelder V.
Published: April 2009

The effect of recombinant hirudin (r-hirudin, LU 52369) and unfractionated heparin on recombinant tissue-type plasminogen activator (rt-PA; LU 50232) thrombolysis and reocclusion rates after successful thrombolysis was studied in a copper-coil-induced thrombus model in the iliac artery of anesthetized rabbits. Simultaneous administration of rt-PA and recombinant hirudin (r-hirudin) at a dose not affecting the partial thromboplastin time (PTT) increased the number of recanalized arteries significantly. The incidence of reocclusion was drastically reduced from 100% to less than 25%. At a dose increasing PTT twofold, unfractionated heparin had no effect on the incidence of reperfusion and reocclusion. These experimental data indicate that the combination of rt-PA with low-dose r-hirudin may be useful for the prevention of early reocclusion in thrombolytic therapy.
Comparative Study of Three Recombinant Hirudins with Heparin in an Experimental Venous Thrombosis Model
Authors: Doutremepuich C. , Lalanne M.C. , Doutremepuich F. , Walenga J. , Fareed J. , Breddin H.K.
Published: April 2009

Three recombinant hirudins (r-hirudins) produced by genetic processes from Escherichia coli and yeast were studied. r-Hirudins could be an alternative treatment to heparin; so, the antithrombotic activity of these drugs should be compared to heparin, the reference substance, in an experimental venous thrombosis model. In this model, the effect of these r-hirudins on thrombus weight reduction were not identical. They varied depending on the original product (E. coli or yeast). The growth-inhibiting activity of r-hirudins on existing thrombi is not dose dependent, whereas that of heparin is. Moreover, in the conditions of this study, higher doses of heparin, but not of hirudins, increased the bleeding time. Although hirudin has limited applications for the time being, it seems an interesting anticoagulant drug, and the availability of r-hirudin opens new therapeutic anticoagulation perspectives.
The Effects of Recombinant Hirudin on Various Platelet Functions
Authors: Basic-Micic M. , Rauschenbach Ch. , Breddin H.K.
Published: April 2009

The effect of two recombinant hirudins, HBW 023 (Hoechst AG, Frankfurt, FRG) and CGP 39393 [recy(tyr 63)] (Ciba Geigy, Basel, Switzerland) on various platelet functions has been investigated in blood samples of healthy volunteers anticoagulated with hirudin at concentrations of 10, 30, 50 μg/ml blood. Spontaneous aggregation was rarely observed in hirudinized platelet-rich plasma (PRP) compared to citrate-PRP. In comparison with citrate-PRP, both hirudins reduced the maximal response to epinephrine, but had no influence on the maximal response to ADP. Both hirudins reduced platelet adhesion to siliconized glass and inhibited platelet spreading compared to citrate-PRP. If hirudin was added to citrate-PRP, platelet count, epinephrine-induced aggregation, platelet spreading and adhesion remained unchanged while the maximal response to ADP increased. If citrate was added to hirudin-PRP, the maximal response to epinephrine as well as platelet spreading and adhesion increased to the extent observed in citrate-PRP. The effects of hirudin on platelet function are more pronounced than those of unfractionated heparin.
Platelet Aggregation in Recombinant-Hirudin-Anticoagulated Blood
Authors: Glusa E.
Published: April 2009

Recombinant hirudin (r-hirudin) inhibited the thrombin-induced aggregation and 14C-serotonin secretion of human platelets in the same concentration range as native hirudin. In r-hirudinized blood, lower spontaneous platelet aggregation was found than in citrated or heparinized blood. Except for a demonstrable aggregation-potentiating effect, adrenaline did not induce aggregation in r-hirudin- or in citrate-anticoagulated blood. The lowest platelet adhesion to glass surface was found in r-hirudinized plasma. The ADP-induced aggregation was nearly the same in the three differently anticoagulated plasma samples: however, desaggregation predominated in r-hirudinized plasma. Adrenaline caused only a slight aggregation in r-hirudinized plasma. ADP and adrenaline caused 14C-serotonin secretion in citrated plasma only. The collagen- as well as the PAF- and arachidonic-acid-induced aggregation did not differ significantly in the three plasma samples. Hence, r-hirudin is suitable for studying platelet functions at physiological calcium concentrations.
Antithrombotic Action of Recombinant Hirudin in a Venous Thrombosis Model
Authors: Raake W. , Klauser R.J. , Elling H. , Zeiller P.
Published: April 2009

The dose- and time-dependent antithrombotic activity of recombinant hirudin (r-hirudin) was examined in a new animal model of venous thrombosis. For the evaluation of the antithrombotic activity of r-hirudin, an occluding thrombus was produced by repeated clamping of the rat jugular vein. The number of clampings necessary to induce thrombosis served as a measure of antithrombotic activity. The gradual reduction of blood flow was registered by Doppler sonography. In order to estimate the relative antithrombotic activity of r-hirudin, its potency in this model was compared with other antithrombotic substances. r-Hirudin proved to be less potent than heparin after intravenous administration. Its half-life after subcutaneous administration was relatively short; however, the antithrombotic potency after subcutaneous administration was comparable to that of heparin.
Clinical Pharmacology of Recombinant Hirudin
Authors: Markwardt F. , Nowak G. , Stürzebecher J.
Published: April 2009

Pharmacological profiling of recombinant hirudin (r-hirudin) has shown that this selective tight-binding thrombin inhibitor is a potent, well-tolerated anticoagulant. Clinical pharmacological studies were performed in human volunteers after single and repeated doses of 0.1–0.5 mg/kg. Generally, administration of r-hirudin was tolerated without side effects. Thrombin time and partial thromboplastin time were prolonged dependent on the r-hirudin level in plasma. Platelet counts, fibrinogen level and fibrinolytic system remained unchanged. Bleeding time was not prolonged. On intravenous injection, r-hirudin was rapidly distributed into the extracellular space and eliminated, with a dose-dependent half-life of 1–2 h (first-order kinetics). After subcutaneous administration, the rH level in blood reached plateau values within 60–120 min. The high recovery of unchanged r-hirudin in the urine identified renal excretion as the predominant route of r-hirudin clearance.
Hirudin as Anticoagulant in Experimental Hemodialysis
Authors: Markwardt F. , Nowak G. , Bucha E.
Published: April 2009

After genetically engineered recombinant DNA desulfatohirudin (r-hirudin) had been investigated as to its pharmacokinetic behavior and blood level course in nephrectomized dogs, the compound was studied for its capability to prevent thrombus formation in the extracorporeal circulation. Beagle dogs underwent bilateral functional nephrectomy followed by experimental hemodialysis. r-Hirudin content in blood, fibrinogen level as well as platelet count were determined before and during the dialysis. Furthermore, the blood loss during the experiment was measured as well as the mean arterial pressure and the pressure in the blood line system. Intravenous administration of the thrombin inhibitor resulted in initial distribution in the extracellular space (distribution phase 90 min) followed by retarded decrease of the r-hirudin blood level (t½β ∼ 6–8 h) which is due to the missing renal excretion of the inhibitor. This caused a long-lasting, dose-dependent anticoagulant effect, which is not only characterized by the prevention of increasing pressure before the capillary dialyzer but also by the reduced drop in fibrinogen and platelets during hemodialysis. The required dose of r-hirudin (0.5 mg/kg) is within a range where bleeding complications will not occur yet.
Hirudin – A Potential Stabilizing Factor for Platelet Preservation in Transfusion
Authors: Pindur G. , Wenzel E. , Koehler M. , Heiden M. , Braun B.
Published: April 2009

The influence of additional hirudin in low doses on platelet and coagulation parameters was studied in platelet concentrates (PC) prepared by cytapheresis using citrate-dextrose solution formula A. The in vitro release of platelet factor 4 and β-thromboglobulin was markedly lowered and morphological platelet alterations, as assessed by platelet size studies were considerably less accentuated in the presence of hirudin over a 5-day storage period; this suggests a possibly protecting effect on platelet function. No clear activation of the coagulation and fibrinolysis system was evident during PC storage with and without hirudin since thrombin-antithrombin III complexes and D-dimers did not markedly change.
Disruption of SEMA4D Ameliorates Platelet Hypersensitivity in Dyslipidemia and Confers Protection Against the Development of Atherosclerosis
Authors: Zhu Li , Stalker Timothy J. , Fong Karen P. , Jiang Hong , Tran Anh , Crichton Irene , Lee Eric K. , Neeves Keith B. , Maloney Sean F. , Kikutani Hitoshi , Kumanogoh Atsushi , Pure Ellen , Diamond Scott L. , Brass Lawrence F.
Published: April 2009

Objective—

In dyslipidemic states, platelets become hyperreactive, secreting molecules that promote atherosclerosis. We have shown that the semaphorin family member, sema4D (CD100), is expressed on the surface of platelets and proposed that its role includes promoting thrombus growth by binding to nearby platelets and endothelial cells, both of which express sema4D receptors. Here we tested the hypothesis that deleting sema4D will attenuate the adverse consequences of dyslipidemia on platelets and the vessel wall.

Methods and Results—

Platelet function and atherosclerotic lesion formation were measured in LDLR(−/−) and sema4D(−/−)LDLR(−/−) mice after 6 months on a high-fat diet. All of the mice developed the dyslipidemia expected on this diet in the absence of functional LDL receptors. However, when compared to LDLR(−/−) mice, sema4D(−/−) LDLR(−/−) mice had reduced lipid deposition in the descending aorta, a 6-fold decrease in the frequency of arterial occlusion and a reduction to near wild-type levels in the accumulation of platelets after injury. These differences were retained ex vivo, with a marked decrease in platelet accumulation on collagen under flow and in platelet aggregation.

Conclusions—

These results show that loss of sema4D expression reduces the platelet hyperactivity otherwise found in dyslipidemia, and confers protection against the development of atherosclerosis.