bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Plasma Alpha-Granule Membrane Protein GMP-140 Using Radiolabeled Monoclonal Antibodies in Thrombotic Diseases
Authors: Wu Guoxin , Li Fugang , Li Peixia , Ruan Changgeng
Published: April 2009

There is an assumption that platelet activation and endothelium damage play a critical role in the pathogenesis of thrombotic disorders. A radioimmunoassay based on using two monoclonal antibodies (MAbs) to different epitopes of α-granule membrane protein (GMP-140) was used to determine whether plasma GMP-140 can be detected in patients suffering from acute myocardial infarction (AMI) or cerebral thrombosis and in patients during cardiopulmonary bypass (CPB). MAb SZ-51 was used as a solid phase, and 125I-labeled MAb S12 was used as a fluid phase. The assay is so sensitive that it can detect as little as 1 ng/ml of purified GMP-140. The intra- and interassay coefficients of variation were 4.2% (n = 5) and 7.1 % (n = 8), respectively. The concentration of plasma GMP-140 was found to be 10.0 ± 4.5 ng/ml (mean ± SD, n = 20) in normal subjects. Ten patients undergoing CPB demonstrated a transient increase in the concentration of plasma GMP-140, especially at 2 h after CPB, and the plasma GMP-140 level was inversely correlated with the decreased platelet counts during bypass (r = -0.81, p < 0.01). It was found that the concentration of plasma GMP-140 increased significantly after AMI. Plasma GMP-140 reached the peak within 3 days and changed with the process of AMI (n = 16) patients. The concentration of plasma GMP-140 increased significantly in patients with cerebral thrombosis in the acute phase but not after relief. These data considered together suggest that plasma GMP-140 can be reliably detected by radioimmunoassay based on using two MAbs which can recognize the different epitopes of GMP-140, and plasma GMP-140 may provide a useful marker for thrombosis and some thrombotic diseases.
Coagulation, Fibrinolytic and Kallikrein Systems in Neonates with Uncomplicated Sepsis and Septic Shock
Authors: Roman J. , Velasco F. , Fernandez F. , Fernandez M. , Villalba R. , Rubio V. , Vicente A. , Torres A.
Published: April 2009

This study evaluates the contact system, coagulation inhibitors and fibrinolysis in 23 full-term newborns with sepsis (8 with septic shock). The results were compared with a group of 20 healthy newborns. Blood samples were obtained at the time of clinical diagnosis and 3 days after the antibiotic therapy was started. The results showed that: severe infection was associated with activation of the contact system, depletion of anticoagulant proteins and elevation of C4b-binding protein levels. There was a shift in protein S to the complexed inactive form, and the thrombin-antithrombin complexes increased. These changes occurred in parallel to both activation and inhibition of fibrinolysis. These changes were more pronounced in the septic shock patients than in nonshock neonates. After therapy, this procoagulant state decreased among survivor patients while in those who died, the abnormalities in coagulation did not improve. Our study suggests that neonatal sepsis induces a hypercoagulable state that persists in nonsurvivor neonates despite a correct treatment.
Reduced Fibrinolytic Activity in Glomeruli Isolated from Rabbits Infused with Tumor Necrosis Factor
Authors: Colucci M. , Zoja C. , Remuzzi G. , Semeraro N.
Published: April 2009

Glomerular fibrin deposition may result from local activation of blood coagulation and/or impaired removal by the fibrinolytic system. We evaluated the fibrinolytic activity of glomeruli isolated from rabbits infused for 5 h with tumor necrosis factor (TNF) at the doses of 0.8 μg/kg/h (n = 5) and 8 μg/kg/h (n = 3) or with endotoxin (8 μg/kg/h, n = 7). Animals infused with vehicle (n = 11) served as control group. Plasminogen activator (PA) activity of glomerular extracts from rabbits infused with 8 μg/kg/h of TNF or endotoxin was significantly lower than that of samples from control animals (p = 0.013 and p = 0.003, respectively). At the dose of 0.8 μg/kg/h, TNF caused a reduction in glomerular PA activity which, however, did not reach statistical significance. Neither plasminogen-independent activity nor PA inhibitor activity was detected in glomerular extracts. Fibrin autography of control extracts revealed the presence of two main fibrinolytic activities comigrating with purified urokinase-type and tissue-type PAs. In treated samples, the bands corresponding to free PAs were markedly reduced, with no evidence of enzyme-inhibitor complex formation. Local reduction of fibrinolytic capacity may contribute to intraglomerular fibrin deposition during endotoxemia or in renal inflammatory diseases associated with TNF production.
Successful Administration of Low Molecular Weight Heparin in a Patient with Heparin-lnduced Thrombocytopenia and Coumarin-lnduced Skin Necrosis
Authors: Drakos P. , Uziely B. , Nagler A. , Gillis S. , Eldor A.
Published: April 2009

A case of heparin-induced thrombocytopenia and skin necrosis induced by coumarin is reported in a 58-year-old female patient suffering from metastatic breast adenocarcinoma and deep vein thrombosis. The thrombosis resolved after treatment with low molecular weight heparin and the thrombocytopenia recovered within a few days. The occurrence of both these rare complications in the same patient has not been previously reported in the literature. We present a review of these complications and their current treatment.
Flow-Cytometric Analysis of Mepacrine-Labelled Platelets in Patients with End-Stage Renal Failure
Authors: Gawaz Meinrad P. , Bogner Caroline , Gurland Hans J.
Published: April 2009

Platelet dysfunction and increased bleeding tendency has been the most consistently described haemostatic abnormality in patients with renal failure. Besides abnormalities in platelet membrane glycoproteins, a reduced amount of platelet-dense granule content has been demonstrated in patients with end-stage renal failure (ESRD) indicating an acquired storage pool deficiency (SPD) present in uraemia. To study dense granules, platelets were labelled with mepacrine, a fluorescent probe which is specifically incorporated into dense bodies. MepaPlatelets of 13 patients with ESRD and of 11 healthy controls were studied. The results showed that mepacrine-labelled platelets of patients with ESRD reveal a significantly (p < 0.05) reduced fluorescence compared to the control group. This implies a reduced number or content of dense granules present in ESRD platelets. Thus, the current data indicate that ESRD is associated with an acquired platelet SPD which may play an important role in the pathophysiology of uraemic bleeding tendency. Moreover, the described FACS assay may be a useful and rapid method for screening patients with suspected acquired or inherited SPD.
Venous Duplex Imaging for the Diagnosis of Acute Deep Venous Thrombosis
Authors: Comerota Anthony J. , Katz Mira L. , Hashemi Homayoun A.
Published: April 2009

Acute deep venous thrombosis (DVT) continues to be a common clinical problem requiring objective evaluation. Hemo-dynamic testing for acute DVT has been popular, but is inadequate for evaluating asymptomatic patients and symptomatic patients with isolated calf vein thrombi. Venous duplex imaging (VDI) has rapidly gained in popularity, and is generally accepted to be the noninvasive technique of choice for the evaluation of patients with acute DVT. Twenty-five reports evaluate gray-scale venous duplex imaging versus ascending phlebography in 2,781 symptomatic patients. The sensitivity for proximal DVT and calf DVT is 96 and 80%, respectively. Seven reports review the use of VDI for surveillance in 857 asymptomatic patients, with an overall sensitivity of 76% for proximal DVT and of 11 % for isolated calf vein thrombosis. The results of color-flow duplex appear to be somewhat better; however, the numbers are considerably smaller. The results for identification of calf vein thrombosis in asymptomatic surveillance patients continue to be poor. VDI appears to be the best noninvasive diagnostic test for acute DVT, and may challenge ascending phlebography as the best diagnostic test for proximal DVT in symptomatic patients, although it will miss 20% of isolated calf DVT. VDI appears to be the best noninvasive screening technique for high-risk asymptomatic patients under surveillance; however, additional correlative studies with ascending phlebography are required. The addition of color Doppler images appears to have improved results, although these higher sensitivities may be the consequence of improved experience as much as the addition of color to the image.
Venography in Deep Venous Thrombosis: Postoperative Screening of Patients in Prophylaxis Studies
Authors: Borris Lars C. , Lassen Michael R.
Published: April 2009

Venography is the key diagnostic method of screening for deep venous thrombosis in clinical prophylaxis studies. Although the venographic technique is standardized the outcome is subject to variation for several reasons. The different methodological pitfalls are presented as well as their consequences for the final results of prophylaxis studies, and it is discussed how variations can be minimized.
Comments on Reason for Conflicting Results of Venography
Authors: Nylander Göran
Published: April 2009

Conflicting results of phlebography in the diagnosis of thrombosis in different investigations are discussed. The following factors may explain different results: phlebography is a subjective method, not based on measurable parameters; insufficient phlebographic technique may result in unsafe diagnosis; the selection of patient material may vary in different investigations; variations in thrombosis incidence may vary with the age of the patient and with the season; the incidence of thrombosis varies within the postoperative period.
Neutrophils and Deep Venous Thrombosis
Authors: Stewart Gwendolyn J.
Published: April 2009

A case can be made for the participation of polymorphonu-clears (PMN) in the initiation and propagation of venous thrombosis. In animal models leukocytes adhered to areas of veins that serve as sites for initiation of thrombi in patients. In addition, PMN are found in white layers of thrombin where they may interact with platelets to attract more of each. This would add bulk and promote coagulation so that red layers are formed. Lidocaine and one of its derivatives inhibited leukocyte adhesion to veins in dogs and lidocaine reduced the incidence of deep venous thrombosis (DVT) in patients after hip replacement, suggesting but not proving that inhibition of PMN adhesion might have contributed. A new approach for preventing PMN contribution to DVT is suggested by recent studies wich identified three families of adhesive receptors (integrins, intercellular adhesion molecules and selectins) on endothelium, leukocytes and platelets. Monoclonal antibodies against β2-integrins on leukocytes reduced leukocyte adhesion, emigration and PMN-dependent tissue injury in infection, inflammation and ischemia-reperfusion injury in animals. Selectins bind to specific carbohydrate ligands containing sialylated Lewis X, suggesting that relatively small analogues might inhibit PMN adhesion. Both platelets and PMN adhere to polymerizing fibrin through undefined mechanisms. Inhibition of this process might inhibit the buildup of white layers of thrombi.
Thrombin in Arterial Thrombosis
Authors: Badimon Lina , Meyer Beat J. , Badimon Juan J.
Published: April 2009

Thrombin plays a pivotal role in the pathogenesis of arterial thrombosis. Platelet-rich thrombosis after arterial injury is dependent on the depth and extent of injury, local Theological conditions and the persistence of residual mural thrombus. The unique effects of specific thrombin inhibitors, including thrombin action on platelets and blood coagulation, demonstrated in experimental and preliminary clinical trials, suggest that specific thrombin inhibitors may be the next major advance in antithrombotic therapy of acute coronary syndromes. The importance of thrombin lies not just in acute thrombus formation following arterial injury but thrombin also contributes to smooth muscle cell proliferation by stimulating platelet secretion of growth factors (especially platelet derived growth factor, PDGF) and directly acting on smooth muscle cells. Thus, thrombin has direct effects on cell proliferation and influences the cellular synthetic mechanisms responsible for matrix protein and collagen production. The role for thrombin as a possible mitogen for vascular cells has gained support by the identification of a cellular thrombin receptor and by the recent detection of m-RNA for this receptor in human atherosclerotic plaques. Therefore, specific thrombin inhibition may also have a potential impact on the relative proliferative response of endothelial and smooth muscle cells after arterial injury such as PTCA by preventing restenosis.
Inherited Resistance to Activated Protein C, a Major Cause of Venous Thrombosis, is due to a Mutation in the Factor V Gene
Authors: Dahlbäck B.
Published: April 2009

Our laboratory recently found a novel mechanism for thrombophilia, which is characterized by an inherited resistance to activated protein C (APC). The APC-resistance test, which measures the anticoagulant response to APC in an activated partial thromboplasin time (APTT) reaction, was devised and used to screen a cohort of consecutive thrombosis patients. APC-resistance was found in approximately 40% of the cases. Other known causes for thrombosis, such as deficiencies of protein C, protein S or antithrombin, were found in another 5% of the patients. Our results, which have recently been confirmed from other laboratories, suggest APC-resistance to be highly prevalent in thrombosis patients. In a majority of cases, APC-resistance was demonstrated to be inherited and family studies revealed an autosomal dominant mode of inheritance. In the investigated families, APC-resistance was associated with thrombosis, which suggests a causal relationship between APC-resistance and thrombosis. An anticoagulant cofactor activity, which corrected APC-resistance, was found in normal plasma, whereas plasma from an individual with pronounced APC-resistance was devoid of this activity. Purification and characterization of the novel APC-cofactor suprprisingly revealed that it was identical to coagulation factor V. Thus, factor V is not only an important procoagulant, but also expresses anticoagulant properties as a cofactor to APC. Our present data suggest the anticoagulant function to be a property of unactivated factor V, whereas the procoagulant activity is expressed after activation to Va. APC-resistant individuals have normal levels of procoagulant V-activity, it is now known that APC-resistance is caused by mutation in the factor V gene changing arginine 506 to a glutamine, thus affecting the APC-cleavage site.
Plasma Product Treatment in Various Types of von Willebrand’s Disease
Authors: Berntorp Erik
Published: April 2009

Four different virus-inactivated factor VIII concentrates (Haemate P, Behring; Profilate, Alpha, FVIII-VHP-vWF, CRTS), near-pure von Willebrand factor (Facteur Willebrand, CRTS) or one recombinant FVIII preparation (Recombinate, Baxter) were given to one or more patients with different forms of von Willebrand’s disease. Duke bleeding time, VIII:C, vWF:Ag, RCof activity, and the multimeric pattern of plasma vWF were monitored. Both Duke bleeding time and the multimeric pattern were normalized after treatment with Haemate P, FVIII-VHP-vWF, or Facteur Willebrand, and to a lesser extent after Profilate. Except in one case, the reduction in bleeding time lasted longer after Haemate P than after the other concentrates. Recombinate had no effect on primary hemostasis, and the half-life of VIII:C was very short. If prompt hemostasis is required, and when pharmacological correction of the defect is impossible, we recommend a concentrate containing both FVIII and the full complement of vWF multimers, but for prophylactic treatment pure von Willebrand factor may be used.
Noninvasive Objective Tests for the Diagnosis of Clinically Suspected Deep-Vein Thrombosis
Authors: Cogo Alberto , Lensing Anthonie W.A. , Wells Phil , Prandoni Paolo , Büller Harry R.
Published: April 2009

Deep-vein thrombosis of the lower extremity is a frequent disorder associated with morbidity and mortality due to pulmonary embolism and the postthrombotic syndrome. It was not until the introduction of contrast venography that the inaccuracy of the clinical diagnosis became apparent. Since then, management decisions have usually been based on objective diagnostic test. Venography is generally considered the reference method for the diagnosis of deep-vein thrombosis, but it is invasive and associated with serious side effects. Several noninvasive or less invasive objective diagnostic methods have been developed. These diagnostic methods are distinctly different in technology and consequently in their ability to demonstrate or refute deep-vein thrombosis. In this review, a critical analysis is provided on the accurary of the current noninvasive diagnostic approaches to venous thrombosis in patients with a first episode of clinically suspected deep-vein thrombosis. Results of studies were considered only when their methodology fulfilled the essential criteria for evaluation of a diagnostic test.
Failure of the Synthetic Androgen, Danazol, to Increase Protein S Levels and Prevent Thrombosis in a Patient with Severe Protein S Deficiency
Authors: Bower William A. , Humphries John E.
Published: April 2009

A 34-year-old man with recurrent deep and superficial thromboses was found to have severe protein S deficiency. Treatment with both warfarin and adjusted-dose subcutaneous heparin failed to completely prevent thrombosis. Based on reports of increases in the endogenous anticoagulants (protein C, protein S, antithrombin III and plasminogen) with synthetic androgen therapy, the patient was treated with danazol for 8 weeks. Although the levels of antithrombin III, protein C and plasminogen increased, no change in the levels of total or free protein S or C4b binding protein was observed. Treatment was discontinued at 8 weeks when the patient developed a recurrence of superficial thrombophlebitis. The role of synthetic androgens in the treatment of patients with inherited thrombotic disorders is reviewed and potential reasons for treatment failure in this patient are discussed.
Unstimulated and Thrombin-Stimulated Platelets Binding to Immobilized Fibrinogen and Fibrin on Polystyrene Supports
Authors: Martínez-Sales Vicenta , Vila Virtudes , Réganon Edelmiro , Aznar Justo
Published: April 2009

The binding of unstimulated and thrombin-stimulated platelets was studied with immobilized fibrinogen and fibrin on polystyrene. The amount of fibrinogen bound to the polystyrene support was 2 μg/tube, which represents 2.35 μg/cm2. Immobilized fibrin was obtained by adding thrombin (5 nM) to immobilized fibrinogen. The number of unstimulated 111ln-platelets bound to immobilized fibrinogen and fibrin was similar (3.2 ± 0.3 × 106 and 3.1 ± 0.4 × 106 platelets/μg fibrin-(ogen), respectively). The platelet binding steadily increased. In the first 2 min, the binding rate was 0.23 × 106 platelets/μg fibrinogen/min. The binding rate then increased rapidly and saturation was reached at 10 min. The extent of the adhesion of resting platelets to immobilized fibrinogen is about one half that of the same platelets stimulated with thrombin. In thrombin-stimulated 111In-platelets, the binding to immobilized fibrinogen and fibrin is time dependent, and saturation is reached at 5 min. The early rate of thrombin-stimulated platelet binding to fibrinogen is about twice that of binding to fibrin (1.25 and 0.74 × 106 platelets/μg fibrin(ogen)/min, respectively). In saturation conditions, 1 μg fibrinogen binds 5.7 ± 0.6 × 106 thrombin-stimulated platelets and 1 μg fibrin binds 4.6 ± 0.5 × 106 thrombin-stimulated platelets. Our results indicate that the rate of platelet aggregation is faster than fibrin formation, and the rate of fibrinogen-platelet binding is faster than that of fibrin-platelet binding. Therefore, after thrombin stimulation, the binding of platelets to fibrin must be secondary to the binding of platelets to fibrinogen.
Investigation of Activated Prothrombin Complex Concentrate as Potential Hirudin Antidote in Animal Models
Authors: Diehl Karl-Heinz , Römisch Jürgen , Hein Berndt , Jessel Andreas , Ronneberger Hansjörg , Pâques Eric-Paul
Published: April 2009

The specific thrombin inhibitor r-hirudin (HBW 023) has been demonstrated to be effective in preventing thrombosis in preclinical models. Up to now, no bleeding complications have been observed using therapeutically effective doses in animal studies. However, in case of inadvertent overdosing the occurrence of undesired impairment of coagulation cannot be excluded. As a potential antidote an activated prothrombin complex concentrate (APC) was tested on its ability to normalize blood coagulation. APC given as bolus injections 5 min after termination of 1-hour r-hirudin infusions of 0.1, 0.3, 1.0 and 3.0 mg/kg neutralized the r-hirudin-induced prolongation of whole blood coagulation time in rabbits completely within 5 min without any clot formation in the blood vessels or capillaries of the heart, kidneys, or lungs. Furthermore, bleeding time prolongation induced by bolus application of 3.0 and 30.0 mg/kg r-hirudin was significantly inhibited by APC within 5 min. These results suggest that administration of APC may be an effective way to reverse the effects of r-hirudin in the coagulation system in case of inadvertent overdosing of r-hirudin.
The Haemorrhagic Effect of Low Molecular Weight Heparins, Dermatan Sulphate and Hirudin
Authors: Matthíasson Stefán E. , Lindblad Bengt , Stjernquist Ulf , Bergqvist David
Published: April 2009

In a randomized, blind study the primary effect on haemostasis after intravenous administration of dermatan sulphate (DS), recombinant hirudin (r-hirudin) and four commercial low molecular weight heparins (LMWHs) (nadroparine, enoxaparin, dalteparin and tinzaparin) was investigated in rats and compared with saline (control). The tail bleeding time, the bleeding from the gastric mucosa [the mucosal bleeding time (min) and the mucosal bleeding (μ1)] as well as changes in activated partial thromboplastin time, antifactor IIa and Xa activities were investigated. DS and r-hirudin were investigated in a dose potentially suitable in thomboprophylaxis and the LMWHs in doses recommended by the manufacturers for thromboprophylaxis, adjusted to body weight. All substances significantly prolonged the mucosal bleeding time. Dalteparin, tinzaparin, DS and r-hirudin increased the mucosal bleeding when compared with controls whereas nadroparine and enoxaparin did not. The effect of r-hirudin was also significantly more pronounced compared with other treatments. Moreover, r-hirudin prolonged the tail bleeding time significantly whereas the other substances did not. The antifactor Xa activity in plasma correlated well with the given dose of the LMWHs (rs = 0.7). However, the monitored bleeding parameters in the LMWH groups did not correlate with the plasma activities of antifactor IIa or Xa. The results indicate that the tested LMWHs are not equipotent in their effect on haemostasis in this model and that antifactor Ha or Xa activities do not directly correlate with their effect on haemostasis although increased haemorrhage was observed in the LMWHs with lower antifactor Xa/antifactor IIa ratios. Our findings indicate that the different commercial LMWHs may differ in their haemorrhagic effect when given in recommended clinical doses adjusted to body weight. DS (3.0 mg/kg) did not cause more bleeding than the tested LMWHs but r-hirudin (2.0 mg/kg) caused considerable bleeding.
Clottable to Immunological Fibrinogen Ratio in Plasma from Control Subjects and Hyperfibrinogenemic Patients
Authors: Prisco Domenico , Zarone Nicoletta , Liotta Agatina Alessandrello , Cellai Anna P. , Comeglio Paolo , Fedi Sandra , Francalanci Isa , Abbate Rosanna
Published: April 2009

The measurement of fibrinogen (Fg) plasma levels is one of the more frequently performed tests in clinical practice, usually by clotting assay. However, for the diagnosis of dysfibrinogenemia the use of an immunological assay is necessary to compare total and clottable protein. Little information is available on the range of the ratio clottable (C) Fg/immunological (I) Fg levels in normal population. This study aimed at evaluating the CFg/IFg ratio in 70 control subjects (age range 17-74 years – group A), in 57 acute patients (age range 17-79 years – group B) and in 14 pregnant women (age range 27-41 years, pregnancy weeks 30-40 – group C), as a physiologic model of hyperfibrinogenemia. CFg was assayed on citrated plasma by the Clauss clotting method and IFg was assayed by radial immunodiffusion technique. In the three groups, CFg/IFg ratios were not significantly different (respectively group A 0.98 ± 0.17, group B 1.02 ± 0.18 and group C 1.01 ± 0.11), whereas both CFg (310 ± 45 mg/dl) and IFg (326 ± 70mg/dl) levels were lower (p < 0.001) in control subjects than in patients (CFg 556 ± 92 mg/dl; IFg 561 ± 121 mg/dl) and in pregnant women (CFg 530 ± 65 mg/dl; IFg 530 ± 77 mg/dl). The analysis of the relationship between CFg and IFg in the three groups (group A: y = 11.53 + 1.01x, r = 0.64, p < 0.001; group B: y = 68.72 + 0.88x, r = 0.67, p < 0.001; group C: y = 71.59 + 0.87x, r = 0.73, p < 0.01) indicates that a good correlation exists (p < 0.001) for values of fibrinogenemia ranging from 180 to over 700 mg/dl. A reference range of CFg/IFg (mean ± 2 SD in group A) was 0.64-1.32. These data could be of practical importance for a rapid screening of dysfibrinogenemias.
Practical Application of Three Polymorphic Microsatellites in Intron 40 of the Human von Willebrand Factor Gene
Authors: Casaña Pilar , Martinez Francisco , Aznar José Antonio , Lorenzo José Ignacio , Jorquera Juan Ignacio
Published: April 2009

Intron 40 of the human von Willebrand factor gene contains a region with variable-number tandem repeats (VNTR), type (ATCT)n, showing length polymorphism. In order to carry out family studies of von Willebrand’s disease, we performed PCR procedures to analyze 3 previously described microsatellites from that region, both in normal individuals and in von WiUebrand disease patients. Three pairs of primers were used to amplify independently nucleotides 1890-1991 (VNTR 1), 2215-2380 (VNTR 2) and 1640-1794 (VNTR 3) from intron 40. The observed heterozygosities (0.75, 0.73 and 0.86 for VNTRs 1, 2 and 3, respectively) were in good agreement with the expected heterozygosities derived from the allele frequencies (0.70,0.73 and 0.79, respectively). Furthermore, the combination of the 3 VNTRs showed 96% of heterozygosity, which correspond with the 98% expected value under linkage equilibrium. Therefore, our conclusion is that the use of these 3 markers, especially VNTR 3, constitutes a rapid and reliable method for performing segregation studies in von WiUebrand disease families.
Relationship between Plasma Antifactor Xa Activity and the Antithrombotic Activity of Heparins of Different Molecular Mass
Authors: Bianchini P. , Bergonzini G.L. , Parma B. , Osima B.
Published: April 2009

The relationship between the inhibition of venous thrombosis and antifactor Xa (AXa) plasma levels, measured as ex vivo AXa activity at the moment of experimental thrombosis induction, was evaluated in rats treated by different administration routes with different doses of heparins of various molecular masses: unfractionated heparin (UH, 13 kDa), low-molecular-mass heparin (LMM-H, 5kD) and oligo-heparin (OL-H, 2 kD). The AXa activity levels of plasma samples were measured by an amidolytic method and expressed in AXa U/ ml. The antithrombotic effect was determined by a vena cava ligature model and expressed as the percent inhibition of thrombus weight. A correlation between the two parameters was determined, regardless of the administration route used. Every heparin requires different AXa plasma levels to develop the same antithrombotic activity: the plasma concentration inducing a 50% protection was 0.09,0.12 and 0.15 AXa U/ml, for UH, LMM-H and OL-H, respectively. Oligo-H has a significant antithrombotic activity when delivered by the intraileal route and the time course pharmacodynamics showed two phases for the parameters considered: in the second phase, a dissociation between AXa plasma level and antithrombotic effect was observed.
Increased Concentrations of Tumor Necrosis Factor and lnterleukin-6 Contribute to the Hemostatic Abnormalities in Advanced Liver Disease
Authors: Páramo J.A. , Sangro B. , Prósper F. , Quiroga J. , Rifón J. , Rocha E.
Published: April 2009

Abnormal cytokine levels have been described in patients with chronic liver disease, but studies correlating cytokine homeostasis with abnormalities in coagulation and fibrinolysis are lacking. In order to establish a link between cytokines and the hemostatic changes the following parameters were determined in 44 patients with cirrhosis (alcoholic =15, postnecrotic = 22, others = 7): TNF-α, IL·6, thrombin-antithrombin (TAT) complexes, prothrombin fragment 1 + 2 (F1 + 2) and t-PA by using enzyme-linked immunosorbent assays, and PAI-1, plasminogen and α2-antiplasmin (α2-AP) by using chromogenic substrates. All patients were at stages B and C of Child’s classification when entering the study. Mean cytokine concentrations were significantly higher in cirrhotic patients as compared to age- and sex-matched controls (p < 0.009). There was a significant increase of TAT (p < 0.02) and F1 + 2 (p < 0.001) in the patients groups, suggesting a grade of intravascular coagulation. A hyperfibrinolytic state as demonstrated by an increase of t-PA and decrease of plasminogen and α2-AP was also observed (p < 0.001). We could define a subgroup of patients with cytokine values higher than 20 pg/ml. Interestingly, in this group there was a significant increase of TAT (p < 0.04) and t-PA (p < 0.02) levels and a decrease of plasminogen and α2-AP (p < 0.02) as compared to values observed in patients with cytokines lower than 20 pg/ml. We conclude that high levels of TNF-α and IL-6 may contribute to hyperfibrinolysis and intravascular coagulation in patients with liver cirrhosis, as assessed by the increase of TAT and t-PA levels and the reduction of plasminogen and α2-AP.
Protein Z, a New Haemostatic Factor, in Liver Diseases
Authors: Kemkes-Matthes B. , Matthes K.J.
Published: April 2009

Protein Z is a vitamin K-dependent plasma protein, which promotes the association of thrombin with phospholipid surfaces. Deficiency states of protein Z have recently been showr in patients with a bleeding tendency of an otherwise unknowr origin. In the present study severe diminutions of blood plasma protein Z were found in patients with chronic liver diseases. Protein Z levels decreased with increasing severity of liver disease. Strong correlations between protein Z and other plasma proteins of liver origin indicate that protein Z primarily originates from the liver. The cutaneous bleeding tendency in cirrhosis may in part be due to protein Z deficiency.
Influence of Heparin, Protamine and Polybrene on the Time Integral of Thrombin Generation (Endogenous Thrombin Potential)
Authors: Rotteveel R.C. , Roozendaal K.J. , Weijers R.N.M. , Eijsman L.
Published: April 2009

We investigated the anticoagulating and heparin-neutralizing properties of protamine and polybrene (hexadimethrine bromide), using the endogenous thrombin potential (ETP) as the parameter to access plasma coagulability. The hypocoagulability induced by high doses of heparin (3 IU/ml) could be reversed by addition of protamine to a very limited extent only. Polybrene on the other hand did neutralize heparin at the equivalent concentration and a two-fold excess did not influence the ETP parameters. In vivo neutralization of high-dose heparin with protamine should therefore be reconsidered. Our experiments suggest polybrene to be superior over protamine with respect to neutralization of high doses of heparin.
Comparison of a Recombinant Thromboplastin with Thrombotest® for Oral Anticoagulant Control
Authors: Baele G. , Fiers T. , Leroux-Roels G.
Published: April 2009

Thrombotest® results expressed in international normalized ratio (INR) values, obtained in 108 patients on oral anticoagulant treatment, were compared with prothrombin time (PT) results with a recombinant thromboplastin. The former results were obtained on an Amelung coagulometer, the latter on a photo-optical instrument. Using the Thrombotest method, performed within 2 h after sampling as the reference method, a first group of 63 patients had an INR value between 2 and 4. This group was considered as adequately anticoagulated and served as a true positive population in further analysis. The remaining 45 patients (true-negative group) had an INR value below 2 or higher than 4 and could thus be considered as inadequately anticoagulated. Using these definitions, a sensitivity of 86% and a specificity of 96% could be calculated for the PT with the recombinant thromboplastin. All tests from patients on oral anticoagulant treatment were also performed after 24 h storage of the blood or plasma samples at room temperature. When we compared the reference Thrombotest results with those of the late Thrombotest and the late PT recombinant thromboplastin, sensitivities of 86 and 86% as well as specificities of 91 and 96% were found, respectively. In conclusion, PT with a recombinant thromboplastin on a photo-optical instrument, even after prolonged storage of the plasma samples at room temperature, can be considered as suitable for oral anticoagulation control.
Thrombin-Antithrombin III Complexes as an Additional Diagnostic Aid in Pulmonary Embolism
Authors: Carmassi Franco , Morale Marco , De Negri Ferdinando , Puccetti Renzo , Pistelli Francesco , Mariani Giuliano , Pazzagli Marcello , Palla Antonio , Giuntini Carlo
Published: April 2009

Plasma levels of selected coagulation and fibrinolytic parameters (activated partial thromboplastin time, prothrombin time, fibrinogen, antithrombin III, protein C, thrombin-antithrombin III complexes (TAT), plasminogen activator inhibitor-1 (PAI-1), plasminogen, α2-plasmin inhibitor) were evaluated in 90 patients with clinical suspicion of pulmonary embolism (PE). Plasma levels of fibrinogen, PAI-1 and TAT were significantly higher in patients than in controls (p < 0.01): evaluation of TAT displayed a sensitivity of 96.1% and specificity of 30.8%, and positive and negative predictive values of 64.5 and 85.7%, respectively. The number of nonperfused lung segments correlated directly with TAT levels (p < 0.01) and inversely with arterial pO2 values (p < 0.01). No significant difference was found in the other parameters between patients and controls. Our results suggest that the finding of normal TAT plasma levels can help to exclude PE in patients with clinically suspected PE.
Measurement of Transmitted Light as an Indicator of Cryopreserved Platelet Viability
Authors: Arnaud Françoise , Goodrich Raymond P.
Published: April 2009

This study was designed to establish an alternative technique to the morphology scoring method described by Kunicki et al. [Transfusion 1975;15:414-421] using light transmission. A reference scale for platelet quality (expressed as a percentage of fresh cells) was established with a mixture of fresh platelets and damaged platelets frozen without cryoprotectant. The transmittance, read against autologous plasma, was compared to morphology, hypotonic stress response (HSR) and ADP-induced aggregation. The results showed that transmittance, morphology and HSR were related linearly to the percentage of fresh platelets. There was a significant correlation between relative transmittance (RT) and morphology (r = 0.932), and RT and HSR (r = 0.91), but a very poor correlation was found between transmittance and aggregation. Measurements of transmittance provided an estimation of the global aspect of a population of cryopreserved platelets in terms of shape, size and composition, and the relative transmittance of a sample to that of the fresh population could predict morphology and HSR. It is concluded that these measurements offer a valid alternative method to the subjective morphology scoring obtained with cryopreserved platelets.
Can the Haemorrhagic Component of Heparin Be Identified? Or an Attempt at Clean Thinking on a Dirty Drug
Authors: Hemker Coenraad , Béguin Suzette , Kakkar Vijay V.
Published: April 2009

Heparin consists of different classes of molecules. We distinguish below-critical-chain length heparin (BCLM, MW <5,400), with only anti-factor Xa activity and above-critical-chain length material (ACLM, MW > 5,400) with both antithrombin and anti-factor Xa activity. In this article we introduce a division within the ACLM fraction, between extra large material (MW >8,000) and ACLM-low (MW 5,400-8,000). Extra large material is abundantly present in unfractionated heparin but is rare in low-molecular-weight (LMW) heparins. We noted that injection of an LMW heparin causes 5- to 10-fold higher plasma levels of ACLM than injection of a clinically equivalent dose of unfractionated heparin (UFH) and proportionally higher inhibitions of the clotting system. So with LMW heparin one can afford higher levels of anticoagulation than with UFH at a lower risk of bleeding. We surmise that this is caused by the virtual absence of the (haemorrhagic) extra-large-molecular-weight fraction from LMW heparins. A laboratory artefact, i.e. the absence of Ca2+ in the anti-factor Xa tests, makes that heparin mixtures that lack extra large heparin molecules show a (spuriously) high ratio of anti-factor Xa activity over anti-thrombin activity. So the correlation between a high aXa/alla ratio and a favourable ratio of antithrombotic effect over bleeding is not necessarily caused by the presence of BCLM. In fact BCLM is a poor anticoagulant; in mixtures of ACLM and BCLM, ACLM causes by far the larger part of the anticoagulant effect. We surmise that the LMW fraction of ACLM is the active anticoagulant component in any heparin preparation and, isolated, would make a proper third-generation heparin.
Preoperative Platelet Count and Postoperative Blood Loss in Patients Undergoing Hip Surgery: An Inverse Correlation
Authors: Monreal Manuel , Lafoz Elena , Llamazares Jaume , Roncales Javier , Roca Jaume , Granero Xavier
Published: April 2009

In a previous study we tried to assess the clinical usefulness of platelet count (PIC) to confirm whether postoperative pulmonary embolism could be suspected early. Unexpectedly, the 19 patients who subsequently developed pulmonary embolism had significantly lower mean PIC levels even before surgery. In an attempt to discover whether the preoperative PIC levels were associated with a different incidence of postoperative blood loss, we decided to retrospectively study the relationship between preoperative PIC levels and the consequences of blood loss. There were 459 consecutive patients undergoing hip surgery. After excluding 5 patients who died during the first 3 postoperative days, and 16 patients who bled from a definitive anatomic site, there were 438 patients. Blood loss was considered to be excessive when two or more of the following conditions were present: (1) total transfusion requirements exceeding 1,000 ml whole blood or 2 units of packed red cells; (2) a drop in hemoglobin level of 5 g/dl or more, and (3) a hemoglobin level below 8 g/dl at any moment during the first 8 postoperative days. Blood loss was considered to be excessive in 91 patients. Preoperative PIC levels were significantly lower in these patients as compared to patients without the condition (204 ± 52 vs. 236 ± 79 × 109 liter–1; p = 0.0002). When patients were classified according to the quartiles of preoperative PIC, the odds ratio of developing excessive blood loss was 0.69 (95% CI: 0.38-1.26) in patients in the second quartile; 0.57 (95% CI: 0.30-1.06) in the third quartile, and 0.27 (95% CI: 0.13-0.57) in patients in the highest quartile. After adjusting for age, sex, type of surgery and type of prophylaxis, the preoperative PIC levels maintained a statistically significant inverse correlation with postoperative blood loss.
Protamine Sulphate Inhibits Pentasaccharide (SR 80027)-Induced Bleeding without Affecting Its Antithrombotic and Anti-Factor Xa Activity in the Rat
Authors: Bernat André , Herbert Jean-Marc
Published: April 2009

The neutralization of a potent anti-factor Xa pentasaccharide (SR 80027) and heparin-induced bleeding enhancement by protamine sulphate was studied in vivo. Bleeding time, as measured by transection of the tail of anaesthetized rats, increased after the administration of standard heparin and SR 80027. Doses of 0.6 and 2.5 mg/kg of heparin and SR 80027, respectively, were required to enhance blood loss to the same extent (6-fold increase). Protamine sulphate (10 mg/kg, i.v.) reduced blood loss induced by both compounds but also neutralized the anti-factor Xa activity as well as the antithrombotic activity of standard heparin measured in a venous thrombosis model. However, protamine sulphate did not affect the anti-factor Xa activity or the antithrombotic activity of SR 80027. These data suggest that protamine sulphate may be an effective antidote for the bleeding side-effects of SR 80027 but they also indicate that the bleeding tendency associated with this type of compounds cannot be attributed to their anti-factor Xa activity.
Highly Substituted Hydroxyethyl Starch (HES 200/0.62) Leads to Type-l von Willebrand Syndrome after Repeated Administration
Authors: Treib Johannes , Haass Anton , Pindur Gerhard , Miyachita C. , Grauer Markus T. , Jung Friedel , Wenzel Ernst , Schimrigk Klaus
Published: April 2009

Hydroxyethyl starch (HES) is a frequently used plasma substitute that is popular due to a high degree of therapeutic safety. However, the administration of large volumes of highly substituted, high-molecular-weight starch often leads to iatrogenic von Willebrand syndrome (vWS) with hemorrhagic complications. In patients with cerebral circulatory disturbances we carried out hemodilution therapy during 9-10 days, infusing HES 200/0.62. A von Willebrand factor (vWF) multimeric analysis was carried out in 6 patients using a modified western blot according to the sodium dodecyl sulfate agarose gel electrophoresis method. The vWF multimeric analysis showed that all multimers decreased to the same degree, corresponding to type-I vWS.
Reexamination of the Extent of the Activation of Lys78 Plasminogen by Tissue Plasminogen Activator in the Presence of Polymerized Fibrin
Authors: Urano Tetsumei , Takada Yumiko , Ihara Hayato , Takada Akikazu
Published: April 2009

The rate of the conversion of Lys78 plasminogen (Lys78-plg) to Lys78 plasmin by tissue plasminogen activator (tPA) was directly quantitated by SDS-PAGE. The apparent second-order rate constant (Kapp) was (0.27 ± 0.07) × 103/M/s for single-chain tPA and Lys78-plg, and was enhanced approximately 5-fold by fibrinogen and 17-fold by polymerized fibrin. Kapp was (1.57 ± 0.46) × 103/M/s for two-chain tPA and Lys78-plg, and was enhanced approximately 2.6-fold by fibrinogen and 3.6-fold by fibrin. The factor of the enhancement by polymerized fibrin was far less than in previous reports in which chro-mogenic substrate was commonly employed.
Bleeding Complications with New Antithrombotics Used in Ischaemic Heart Disease
Authors: Adgey A.A.J.
Published: April 2009

Despite adjunctive therapy with heparin and aspirin, patients undergoing percutaneous transluminal coronary angioplasty (PTCA) continue to be at risk of abrupt vessel closure and acute ischaemic events. In an attempt to overcome the limitations of traditional antithrombotics, more potent agents have been developed, including direct thrombin inhibitors (e.g., hirudin and hirulog) and new antiplatelet agents [e.g., the gly-coprotein Ilb/IIIa receptor inhibitor c7E3 Fab (ReoProTM)]. Initial phase-III trials of hirudin in patients with acute coronary syndromes identified an excess incidence of major bleeding complications. Some of these trials have been recommenced using lower doses. Reports on phase-III trials of hirulog should be forthcoming soon. Of the new agents, the chimeric monoclonal antibody fragment c7E3 Fab has the most extensive available data. In the phase-III evaluation of 7E3 for the Prevention of Ischemic Complications trial, the administration of a c7E3 Fab bolus plus c7E3 Fab infusion reduced the rate of major ischaemic events by 35% at 30 days (p = 0.008) in patients undergoing high-risk PTCA. Major bleeding episodes occurred more frequently with this regimen than with placebo, although rates of intracranial haemorrhage or surgery for bleeding did not differ between groups. The findings suggest that the risk of bleeding complications might be reduced, without compromising efficacy, by administering heparin on a weight-adjusted basis in patients treated with c7E3 Fab.
Prothrombin Time Standardization by Correction of the Pivka Inhibitor
Authors: Talstad Ingebrigt
Published: April 2009

The standardization of prothrombin time (PT) assays needs two steps: (1) calibration of PT assays towards a reference assay or reference thromboplastin, (2) correction of PT assays according to the calibration. The present recommended calibration by clotting times is favored for the linearity between assays; the clotting times of abnormal plasma are partly prolonged due to the protein induced by vitamin K absence (Pivka) inhibitor. Calibration by coagulation activities also demonstrated linearity between PT assays; the regression line for abnormal plasma deviated from the line of identity due to differences in sensitivity of assays for the Pivka inhibitor. The corrected PT assays demonstrated similar results using calibration by clotting time or coagulation activities, but the correction was simpler for coagulation activities. Patient plasma was the preferable material in calibration by clotting times as well as by coagulation activities. The corrections between the reference assay and other PT assays were equal to the differences in sensitivities for the Pivka inhibitor. The corrected PT assays did not differ from the reference assay by statistical analysis; any of the six PT assays tested might be used as reference assay.
Notes on the Cell Biology of Tissue Factor
Authors: Camerer Eric , Prydz Hans
Published: April 2009

Tissue factor is both a trigger of blood coagulation and a true receptor inducing an intracellular signal upon binding of its ligand factor VII to its extracellular parts. When induced in endothelial cells more than 75% of tissue factor antigen was found on the apical side of the cell.
Plasma Factor VIla: Measurement and Potential Clinical Significance
Authors: Morrissey James H.
Published: April 2009

Factor VIla (FVIIa) is the triggering enzyme of the blood clotting cascade in hemostasis and thrombosis and may play an important role in hypercoagulable states. Indeed, certain epidemiologic studies have found elevated FVII coagulant activity to be an independent risk factor for heart disease. However, FVII circulates as the inert zymogen (FVII), the active enzyme (FVIIa), and possibly other forms as well, obscuring the relationship between any specific form of this enzyme and risk of thrombotic disease. New assay techniques based on soluble mutant tissue factor now permit measurement of FVIIa without interference from the large excess of zymogen FVII in plasma. This in turn has enabled direct examination of the role of plasma FVIIa in disease.
Role of Tissue Factor and Factor VIa in the Coagulant and Inflammatory Response to LD100 Escherichia coliin the Baboon
Authors: Taylor, Jr. Fletcher B.
Published: April 2009

Anti-tissue factor antibody and active site-inhibited factor VIla inhibit both the disseminated intravascular coagulant response to LD100Escherichia coli and its lethal inflammatory effects. In contrast, active site inhibited factor Xa while completely inhibiting disseminated intravascular coagulant does not protect baboons from LD100E. coli. This review examines the role of tissue factor and factor VIla in initiating the disseminated intravascular coagulant response and in amplifying the inflammatory response.