The influence of intrinsic coagulation pathway on blood platelets activation by oxidized cellulose.

Oxidized cellulose is an effective hemostat that works naturally to aid in blood coagulation. The mechanism of its action is not very well understood. Little effect on blood coagulation, but a pronounce decrease in platelet count has been reported upon the addition of the oxidized cellulose to the whole blood. As a marker of platelet activation and aggregation we used serotonin release reaction and turbidity changes in time. We found that oxidized cellulose did not activate washed platelets reconstituted in plasma-free medium or plasma-free medium with fibrinogen; no reduction of platelet count was observed. Serotonin release in platelet-rich plasma incubated with oxidized cellulose started in the range from 5 to 10 min. Serotonin release from platelets reconstituted in plasma deficient in either coagulation factor V, VIII, IX, or XII was delayed. Blood platelets activation by oxidized cellulose requires calcium ions present in dispersion of oxidized cellulose. Factor XIII deficiency had no influence on blood platelets activation by oxidized cellulose. Our results clearly indicate the significance of intrinsic coagulation pathway activation on blood platelets activation by oxidized cellulose and so indirectly on the hemostyptic effect of oxidized cellulose.

[Hereditary form of thrombotic thrombocytopenic purpura]. / Vrozená forma trombotickté trombocytopenické purpury.

BACKGROUND: Thrombotic thrombocytopenic purpura is characterized by microvascular platelet clumping resulting in thrombocytopenia, microangiopathic hemolysis, neurological abnormality, and renal dysfunction. Similar manifestations also occur in patients with the hemolytic uremic syndrome or other types of disorders. Recent studies demonstrate that severe deficiency of the von Willebrand factor cleaving metalloprotease, ADAMTS 13, causes thrombotic thrombocytopenic purpura. Aim of our study was to characterize gene defects causing inherited type of disease. METHODS AND RESULTS: We investigated nine patients with recurrent type of disease with familiar origin and twelve relatives. Samples were taken in a remission of disease. We measured activity of ADAMTS13 (vWF-CP) with modified method of the quantitative immunoblotting of degraded vWF multimers. Mutation screening was carried out by sequencing all 29 exons and flanking intron regions of the ADAMTS13 gene. Five distinct mutations were found. Three of them are novel. CONCLUSIONS: Mutation analysis of the ADAMTS 13 gene brought interesting results in eight patients. We found a one single base frameshift insertion, 4143insA in 8 of 9 unrelated individuals. This investigation represents an advantage in the differential diagnosis of disease since the thrombotic thrombocytopenic purpura phenotype in childhood can be variable and rapid detection of mutation is helpful for the recurrence prevention.

Effects of malondialdehyde content in low density lipoproteins on platelet adhesion.

Platelets play a key role in thrombotic vascular occlusion at the side of an atherosclerotic plaque. Malondialdehyde (MDA) derivatives are components of oxidized LDL. Recent studies suggest enhanced adhesion of platelets on oxidized LDL, but evidence of MDA-modified LDL contribution to enhanced platelet adhesion is missing. The aim of this study was to investigate platelet adhesion on LDLs with various content of MDA and to analyze its physiological relevance. LDLs were isolated by hydroxyapatite chromatography then analyzed. MDA, vitamin E and vitamin A content was estimated by HPLC methods and adhesion of platelets on modified LDLs was assessed. Both MDA and hypochlorite reactions induced an increase of both malondialdehyde content and negative charge in LDLs preparations. In LDLs of iron-overloaded patients both MDA content and negative charge was also significantly increased. The analysis of data showed that enhanced negative charge of LDLs had only negligible influence on platelet adhesion. The binding of platelets on LDL particles modified by MDA resulted in an S-shape curve and was substantially enhanced by high levels of MDA in LDL preparations. Malondialdehyde content both in hypochlorite modified LDLs and in LDLs of iron-overloaded patient matched the lower part of the binding curve. The platelet adhesion on circulating LDLs modified in in vivo conditions of oxidation stress in blood of iron-overloaded patients depends with considerable certainty preferably on non-MDA derived changes in derivatized LDLs. On the other hand, oxidized LDLs in atherosclerotic lesions can be much more extensively modified by MDA resulting in surfaces with enhanced affinity to adhering platelets. Such LDLs may be accessible to platelets, due to endothelial denudation or plaque rupture.

Image analysis of blood platelets adhesion.

Adhesion of blood platelets is one of the major events in haemostatic and thrombotic processes. We studied adhesion of blood platelets on fibrinogen and fibrin dimer sorbed on solid support material (glass, polystyrene). Adhesion was carried on under static and dynamic conditions and measured as percentage of the surface covered with platelets. Within a range of platelet counts in normal and in thrombocytopenic blood we observed a very significant decrease in platelet adhesion on fibrin dimer with bounded active thrombin with decreasing platelet count. Our results show the imperative use of platelet poor blood preparations as control samples in experiments with thrombocytopenic blood. Experiments carried on adhesive surfaces sorbed on polystyrene showed lower relative inaccuracy than on glass. Markedly different behaviour of platelets adhered on the same adhesive surface, which differed only in support material (glass or polystyrene) suggest that adhesion and mainly spreading of platelets depends on physical quality of the surface. While on polystyrene there were no significant differences between fibrin dimer and fibrinogen, adhesion measured on glass support material markedly differed between fibrin dimer and fibrinogen. We compared two methods of thresholding in image analysis of adhered platelets. Results obtained by image analysis of spreaded platelets showed higher relative inaccuracy than results obtained by image analysis of platelets centres and aggregates.

Hemostyptic effect of oxidized cellulose on blood platelets.

Cellulose is one of the hemostyptic biomaterials, which are able to initiate or accelerate blood coagulation at the site of their application. It belongs to surgical sealants. The mechanism of its action is not clearly understood. We studied the participation of blood platelets in this mechanism. As a marker of platelet activation we used serotonin release reaction. Serotonin release in platelet rich plasma incubated with various concentrations of oxidized cellulose (0.5%-2.0%) started in about 20 min. Washed platelets were not directly activated by oxidized cellulose within one hour. Washed platelets reconstituted in plasma obtained from two patients with coagulation factor XII deficiency were activated by oxidized cellulose with a prolonged lag phase. Our results demonstrate the significant influence of factor XII on blood platelets activation by oxidized cellulose.

[Role of free radicals in blood platelet activation]. / Ucast volných radikálu pri aktivaci krevních desticek.

BACKGROUND: Free radicals and reactive oxygen containing substances are in addition to negative effects on biological systems important as signal molecules. Influencing their concentration by the action of antioxidants has a basic influence on the course of a number of cellular responses. The function of platelets is modulated in a significant way by the presence of vitamin E and resveratrol. The objective of the submitted paper is to assess the effect of Trolox (a stable analogue of vitamin E) and resveratrol sorbed by platelets on the aggregation response of washed platelets activated by collagen. METHODS AND RESULTS: To investigate the effects of the mentioned antioxidants on platelet aggregation washed platelets were prepared. The concentrations of the two antioxidants sorbed by platelets were assessed by the method of high performance liquid chromatography. From the total Trolox concentration (4200 microM) the platelets sorbed 33.5 nmol/10(9) platelets and from the total concentration of resveratrol (300 microM) the platelets sorbed 6.5 nmol/10(9) platelets. Inhibited aggregation by collagen was 57% for Trolox and 98% for resveratrol. CONCLUSIONS: The antioxidant capacity of both antioxidants is identical. The resveratrol concentration in platelets which led to almost complete inhibition of platelet aggregation by collagen was five times lower than for Trolox which caused a 57% inhibition of aggregation. Thus also other factors participate in the antioxidant activity of resveratrol. One of these factors is very probably the effect on arachidonic acid cycle.

[Effect of oxidized cellulose on fibrin formation and blood platelets]. / Vliv oxidované celulózy na tvorbu fibrinu a krevní desticky.

BACKGROUND: Haemostyptic materials initiate and hasten blood clotting at the site of their application. The properties of haemostyptic materials are used for treatment of capillary and parenchymatous haemorrhage along with surgical treatment. Celluloses one of the biopolymers studied for a long time, suitable because of its biocompatibility and non-toxicity. METHODS AND RESULTS: In the submitted study the authors used microdispersed calcium-sodium salt of oxidized cellulose which is formed by oxidation of cellulose in position C6 (patent Alltracel Pharmaceuticals). The authors investigated the effect of oxidized cellulose on fibrin formation and platelets. Using the optic method of the surface plasmon resonance they investigated the initial stage of interaction between fibrinogen and oxidized cellulose. Oxidized cellulose retards and reduces the interaction of the immobilized fibrin monomer with fibrinogen. Fibrin formation was investigated spectrophotometrically at 350 nm. In the presence of cellulose the period of formation of fibrin gel was prolonged and its turbidity increased, depending on the concentration of the cellulose used. The platelet activation by cellulose was assessed by measuring the released serotonin. For the activation of platelets by cellulose the presence of plasma is necessary, rinsed platelets were not activated by cellulose. It was revealed that direct, interaction of rinsed platelets or fibrinogen with cellulose plays a secondary role. CONCLUSIONS: These data and the retarded activation of platelets in plasma with factor XII deficiency indicate that due to negatively charged oxidized cellulose probably activation of the contact coagulation system occurs and this leads to the activation of platelets and fibrin formation.

Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography.

Estimation of lipid peroxidation through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products separated by HPLC remains the method of choice to study the development of oxidative stress in blood plasma. In this report we describe the influence of citrate and EDTA anticoagulants used for blood collection on estimation of MDA concentrations using HPLC analysis of MDA-TBA adducts. We analyzed a group of 40 blood donors (21 men and 19 women), median age 27 years, range 19-48 years. The mean MDA concentration in citrate plasma was 1.43+/-0.51 micromol/l (range: 0.61-2.57 micromol/l) and in EDTA plasma 0.36+/-0.10 micromol/l (range: 0.13-0.63 micromol/l). There was a significant difference in MDA mean concentration that we attribute to different antioxidant properties of anticoagulants used for blood collection. Consistency in the choice of anticoagulant is clearly extremely important.

Albumin and heparin multilayer coatings for blood-contacting medical devices.

Three types of covalently crosslinked assemblies consisting of multiple (1) molecular layers of human serum albumin (HSA); (2) alternating layers of HSA and unfractionated heparin; and (3) alternating layers of HSA and partly depolymerized heparin fixed with one end to HSA were prepared on various surfaces. Adsorption of fibrinogen, IgG, and antithrombin (ATIII) from human citrated plasma on coated surfaces was evaluated by ELISA. Fibrinogen adsorption on coated ELISA plates was lower than that on bare polystyrene. There was no IgG adsorption on the HSA coating alone, but considerably high IgG adsorption was detected on the heparin-containing surface. The adsorption of ATIII increased with increasing heparin on the surface. The effect of multilayer coatings on platelets was tested by incubation of modified vascular prostheses with citrated blood. The most favorable interaction with platelets was observed on the HSA assembly. The interaction of platelets with the surface bearing unfractionated heparin was higher than that of the surface covered with partly depolymerized heparin. The long-term durability of the HSA-heparin coating was proven by a 21-day implantation of coated polyurethane plates in goat heart.

Determination of the putative binding sites for thrombin receptor activating peptide through a hydropathic complementary approach.

Putative binding sites in a platelet thrombin receptor (PAR-1) for the activating peptide SFLLRNPNDKYEPF (AP) and for the bradykinin analogue MKRPPGFSPFRSSRIG were revealed using a computer program for identifying complementary peptide segments. The program is based on the assumption that interactions of agonist's peptides and protein's receptors can be elucidated by complementary average hydropathies as much as possible equal by size and opposite by sign. Some of the computer-found putative binding sites were close to the supposed AP-PAR-1 contacts in the amino-terminal exodomain and in the second extracellulary loop of PAR-1. Peptides complementary to these binding sites were also computer-designed and were synthesized. They mostly inhibited the aggregation of gel filtered platelets by thrombin (0.025 U/mL) with IC50 in a high micromolar range of concentrations. The peptide complementary to site L258-Y269 of PAR-1 induced aggregation of gel filtered platelets with EC50 = 98 [micromol/L] related to thrombin (0.025 U/mL) aggregation response.

Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation.

Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600 s(-1) with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s(-1) almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to adsorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.

Solid-phase extraction in malondialdehyde analysis.

Malondialdehyde (MDA) has been widely used as an index of lipoperoxidation in biological and medical sciences as well as in the food industry. A solid-phase extraction (SPE) of the condensation product of the MDA with 2-thiobarbituric acid (TBA) was developed using LiChrolut C18ec, 200 mg (Merck, Darmstadt, Germany), as a SPE cartridge and methanol as an eluent for sample pretreatment before HPLC analysis. The samples of blood plasma, platelet concentrates, or erythrocyte membranes (ghosts) were deproteinized by acetonitrile in the presence of sodium hydroxide prior to the reaction with TBA. The reaction mixture was processed using SPE. The SPE extracts (800 microL of methanol) were put to dryness and after dissolution with 100 microliters of mobile phase, 50 microliters was analyzed by RP-HPLC with fluorescence detection (excitation at 514 nm, emission at 556 nm). The mean MDA concentration in plasmas of 32 healthy donors was 0.37 +/- 0.25 mumol/L and the mean MDA concentration in normal ghosts was 8.3 +/- 4.1 pmol/microgram of protein content. In the case of a patient with a severe form of beta-thalassemia, the concentration of plasma MDA was raised to 1.22 mumol/L and the amount of MDA in erythrocytal ghosts was raised to 21.05 pmol/microgram of protein content. MDA concentration in platelet concentrates (six bags) in the first day of storage was 0.46 +/- 0.18 mumol/L and in the fifth day of storage was 0.55 +/- 0.44 mumol/L.

Simultaneous isolation of protein C activator, fibrin clot promoting enzyme (fibrozyme) and phospholipase A2 from the venom of the southern copperhead snake.

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal-chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A2.

[The Bernard-Soulier syndrome. A hereditary functional and structural disorder of blood platelets]. / Bernarduv-Soulieruv syndrom. Hereditární funkcní a tvarove strukturální porucha krevních desticek.

BACKGROUND: The authors submit a clinical and laboratory description of a patient with Bernard-Soulier syndrome diagnosed in this country for the first time. The investigation comprises an analysis of haemostatic functions, selected structural indicators and the detailed morphology of thrombocytes. The analysis of defined inborn functional disorders of thrombocytes is a method for studying haemostatic mechanisms. METHODS AND RESULTS: In addition to standard haemostatic methods flow cytometry in used to assess the size of platelets, and using monoclonal antibodies against glycoproteins, their presence on the surface membrane is assessed. Thrombocytic glycoproteins are further analyzed using diagonal electrophoresis on sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE). The authors found medium thrombocytopaenia with the presence of giant thrombocytes which on elecronmicroscopic examination, with the occasional exception of endoplasmic membrane proliferation, do not display structural deviations. The assessed deviation of thrombocyte aggregation after ristocetin, suggesting a defect of the adhesive capacity of thrombocytes, is apparently due to reduction of the surface glycoprotein GPIb, proved by flow cytometry and SDS-PAGE. The drop of glycoprotein GPIb to 20% of normal values suggests a heterozygote type of disorder with a medium grade of haemorrhagic manifestations. CONCLUSIONS: This is the first case of Bernard-Soulier syndrome in the Czech literature. Its analysis provides evidence of the relationship of the adhesive function to the surface glycoprotein GPIb and confirm the effectiveness of the elaborated diagnostic methods.

Comparative study of human monocyte and platelet adhesion to hydrogels in vitro - effect of polymer structure.

Blood platelet and monocyte adhesion was studied in vitro with respect to the influence of hydrophilic polymer chemical functional groups and their charge. The results showed that the strongest adhesion of human monocytes was to coverslips covered with cationic polymer. Platelet adhesion to all tested polymers proved to be negligible; no differences related to the charge of the polymers used were observed. These results show the obvious difference between the biocompatibility and haemocompatibility in vitro which must be taken into consideration during polymer biological properties testing before clinical trials.

Comparison of several mouse and rat monoclonal antibodies against human fibrinogen.

Six monoclonal antibodies raised against human fibrinogen have been characterized. Mouse monoclonal antibodies were targeted against sequential epitopes on the immunodominant D-domain of fibrinogen and they crossreacted with all molecules containing the D-domain [fibrin, fibrin(ogen)-degradation products]. Their behavior was not influenced by proteolytic degradation of fibrinogen with plasmin. Rat MoAbs were specific for the conformational epitopes on intact fibrinogen. Their reactivities were substantially lower with fibrin(ogen)-degradation products. Degradation of structures on intact fibrinogen was concomitant with the decay of rat MoAbs reactivity. Those structures were presumably on the C-terminal end of fibrinogen alpha chain and/or on the N-terminal end of fibrinogen beta chain.

Characterization of platelet antigen for CD45RA monoclonal antibodies.

CD45RA monoclonal antibodies recognize the higher molecular weight isoforms (220 and 205 kDa) of leukocyte common antigen family (CD45), which are typically expressed on B cells and unstimulated T cells. We have found that there are at least three distinct CD45RA monoclonal antibodies which react with platelet 42 kDa (P42) intracellular protein antigen, which seems to be different from any to date described platelet proteins with similar molecular weight. This platelet antigen is a single chain protein, very likely not a glykoprotein, with isoelectrical point between 6.8 and 7.5. P42 does not seem to be a membrane protein and is not associated with platelet cytoskeleton. Results of immunofluorescence assay suggest that P42 may be redistributed to platelet surface after platelet aggregation.

Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger.

Using Escherichia coli system expressing papilloma virus HPV16 E7MS2 fusion protein as a model system, a novel procedure was applied to solubilize, purify and refold recombinant proteins from E. coli inclusion bodies. The necessity to reactivate proteins at low protein concentrations (owing to their tendency to aggregate at high concentrations) was overcome by solubilization of inclusion bodies in alkaline solution and immobilization of proteins on a strong and resistant anion exchanger. This procedure has an inherent advantage of combining refolding and purification procedures in one step. The solubilization of the fusion protein in an alkaline reagent with the use of an anion exchanger resulted in considerable purification of the recombinant protein at a fairly high concentration. The protein was soluble under mild conditions and reacted with antibodies against the "native" papilloma virus.

Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-beta-D-thiogalactopyranoside.

The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described. These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions. A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described. The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate. The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves. Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0. The purified protein did not cross-react with antibodies to E. coli.