bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the Urokinase Receptor by Its Plasminogen Activator
Authors: Hollas W , Boyd D
Published: July 2018

There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to assess the role of UK as a modulator of its receptor. GEO colonic cells, which secrete relatively low levels of UK (≃0.1 nM/72 h per 106 cells) and display approximately 104 receptors per cell, 10% of which are "tagged" with the endogenous plasminogen activator (PA), was selected for the study. A 90% reduction in the specific binding of radioactive DFP-UK was observed for cells cultivated in the presence of two-chain (TC) UK (M
r 55,000). This only partly reflected occupation of the receptors with UK supplied in the culture medium, since the specific binding of the radioligand was still reduced by 60% after an acid pretreatment, which dissociates receptor-bound UK. The reduction in radioactive DFP-UK binding to cells treated with high molecular weight UK, either in the single or two-chain form, was both concentration and time dependent. Maximum reductions (70%) were achieved by treatment of the cells for 24 h with 1 nM of the plasminogen activator. In contrast, low molecular weight UK, which lacks part of the UK A chain, had no effect on ligand binding. Attenuation of radioactive DFP-UK binding to UK treated GEO cells was a consequence of a 60% reduction in the number of binding sites. Treatment of GEO cells with an antibody, which blocks the binding of endogenous UK to its receptor, augmented radioactive DFP-UK binding by two-fold. These data indicate that for one colonic cell line, at least, UK down-regulates its own binding site subsequent to it being bound to the receptor.
Improved Adhesion and Proliferation of Human Endothelial Cells on Polyethylene Precoated with Monoclonal Antibodies Directed against Cell Membrane Antigens and Extracellular Matrix Proteins
Authors: Dekker Albert , Poot André A , van Mourik Jan A , Workel Martin P A , Beugeling Tom , Bantjes Adriaan , Feijen Jan , van Aken Willem G
Published: July 2018

Endothelial cell seeding may improve the patency of synthetic vascular grafts provided that platelet reactivity of non-endothelialized sites is not increased. We have investigated if surface-adsorbed monoclonal antibodies directed against endothelial cell membrane proteins and against extracellular matrix proteins promote the adhesion and proliferation of cultured human endothelial cells, without causing platelet deposition at non-endothelialized sites. Adhesion of endothelial cells onto polyethylene coated with monoclonal antibodies directed against endothelial cell-specific membrane antigens, integrin receptors and glycoprotein CD31 was equal to or higher than adhesion onto fibronectin-coated polyethylene. Endothelial cells did not proliferate on these surface-adsorbed antibodies. However, pre-coating of polyethylene with mixtures of endothelial cell-specific monoclonal antibodies and monoclonal antibodies directed against fibronectin or von Willebrand factor, resulted in relatively high adhesion and optimal proliferation. Platelet reactivity of the polyethylene surface was found to significantly increase after adsorption of fibronectin, endothelial cell-specific monoclonal antibody or its Fc fragments. In contrast, adsorption of F(ab')2 fragments of endothelial cell-specific monoclonal antibody did not promote platelet deposition. Therefore, it is concluded that coating of vascular graft materials with mixtures of F(ab')2 fragments of monoclonal antibodies specifically directed against endothelial cells and against extracellular matrix proteins may be an effective way to both promote the growth of seeded endothelial cells and limit platelet-graft interaction.
Comparison of the Accuracy of Impedance Plethysmography and Compression Ultrasonography in Outpatients with Clinically Suspected Deep Vein Thrombosis
Authors: Wells Philip S , Hirsh Jack , Anderson David R , Lensing Anthonie W A , Foster Gary , Kearon Clive , Weitz Jeffrey , Cogo Alberto , Prandoni Paolo , Minuk Terry , Thomson Graham , Benedetti Lino , Girolami Antonio
Published: July 2018

Impedance plethysmography (IPG) and compression ultrasonography (CUS) have been reported to be highly accurate for the diagnosis of deep vein thrombosis (DVT) in symptomatic patients. In many centres CUS has become the method of choice. However, direct comparisons of the accuracy of IPG to CUS have not been performed. To determine the test of choice we performed a two centre prospective comparison of IPG and CUS, with venography, and determined how the size and distribution of thrombi influenced the accuracy of each test. 495 symptomatic outpatients with suspected DVT had evaluable venograms. The prevalence of DVT was 27% (130/495), 84% (109) of which were proximal. The sensitivity of IPG and CUS for proximal vein thrombosis was 77% and 90% respectively (p = .002). The specificity of IPG was 93% whereas the specificity of CUS was 98% (p = 0.04). There were significant differences in accuracy between the two centres as a consequence of differences in the size and location of thrombi. The majority of proximal thrombi not detected by IPG and CUS involved less than 5 cm of the distal half of the popliteal vein and most of these thrombi occurred in one centre. Exclusion of these thrombi from the analysis increases the sensitivity of CUS to 99% (86/87) and IPG to 91% (72/79), for proximal thrombi (p = .019). The positive predictive value of CUS was strongly influenced by the number of abnormal venous segments (three sites were examined); 100% (80/80) if two or three sites were abnormal, but only 68% if a single site was involved. We conclude that: 1) CUS is more accurate than the IPG for the diagnosis of DVT in symptomatic outpatients, and this relationship holds true regardless of the size or location of the DVT, 2) the sensitivities of IPG and CUS are much lower for small proximal DVT, and 3) confirmatory venography is warranted if the abnormality with CUS is limited to one venous segment.
Stimulated Glanzmann’s Thrombasthenia Platelets Produce Microvesicles
Authors: Holme Pål André , Solum Nils Olav , Brosstad Frank , Egberg Nils , Lindahl Tomas L
Published: July 2018

The mechanism of formation of platelet-derived microvesicles remains controversial.

The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann’s thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 μM), and the thrombin receptor agonist peptide SFLLRN (100 μM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 μM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 ±2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 ± 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 μM SFLLRN gave 16.3 ± 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 ± 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.
Endotoxin-Induced Intravascular Coagulation in Rabbits:Effect of Tissue Plasminogen Activator vs Urokinase on PAI Generation, Fibrin Deposits and Mortality
Authors: Paloma M J , Páramo J A , Rocha E
Published: July 2018

We have evaluated the effect of plasminogen activators (t-PA and urokinase) on an experimental model of disseminated intravascular coagulation (DIC) in rabbits by injection of 20 μg/kg/h of E. coli lipopolysaccharide during 6 h t-PA (0.2 mg/kg and 0.7 mg/kg), urokinase (3000 U/kg/h) and saline (control) were given simultaneously with endotoxin. Results indicated that urokinase and low dose of t-PA significantly reduced the increase of plasminogen activator inhibitor (PAI) activity observed 2 h after endotoxin (p <0.001). High t-PA dose also diminished the PAI levels at 6 h (p <0.0001). A significant reduction of fibrin deposits in kidneys was observed in both t-PA treated groups as compared with findings in the group of rabbits infused with saline solution (p <0.005), whereas urokinase had no significant effect on the extent of fibrin deposition. Finally, the mortality rate in the control group (70%) was reduced to 50% in rabbits receiving high doses of t-PA. In conclusion, treatment with t-PA resulted in reduced PAI generation, fibrin deposits and mortality in endotoxin-treated rabbits
Surface Adsorption of Factor XI: Association of Adsorption Sites with the Heavy Chain of Activated Factor XI
Authors: Mannhalter Christine , Schiffman Sandra
Published: July 2018

These experiments study surface adsorption of native and activated factor XI using purified radiolabelled human factor XI and trypsin activated factor XI. Both forms of factor XI adsorb not only to glass but also to polypropylene, polyethylene and polystyrene. Albumin (10 mg/ml) markedly reduces adsorption to plastics but not to glass. Sodium dodecyl sulfate prevents adsorption to all surfaces tested. Reduction of 125I-activated factor XI in the presence of sodium dodecyl sulfate yields labelled chains of molecular weight of about 46,000 (heavy chain), 37,000 (light chain) and a further breakdown product of about 26,000. Reduction of activated factor XI in glass or plastic in the absence of sodium dodecyl sulfate yields only light chain and breakdown product in solution; heavy chain is removed by adsorption. Therefore, we conclude that the adsorption site (s) in trypsin activated factor XI and presumably also in native factor XI is (are) located in the heavy chain subunit of the molecule.
A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq
Authors: Csákó György , Suba Eva A
Published: July 2018

Platelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.

Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.
Stimulated Platelet Aggregation, Thromboxane B2 Formation and Platelet Sensitivity to Prostacyclin - A Critical Evaluation
Authors: Siess Wolfgang , Roth Peter , Weber Peter C
Published: July 2018

Platelets have been implicated in the development of atherosclerotic and thrombotic vascular diseases. Evaluation of platelet aggregation in relation to endogenously formed compounds which affect platelet function may provide information of clinical and pharmacological relevance. We describe a method in which thromboxane B2 (TXB2) formation was analyzed following stimulation of platelet-rich plasma (PRP) with ADP, 1-epinephrine, collagen, and arachidonic acid. In addition, we determined platelet sensitivity to prostacyclin following ADP- and collagen-induced platelet aggregation. The parameters under study were found to depend on the platelet count in PRP, on the type and dose of the aggregating agent used, and on the test time after blood sampling. By standardization of these variables, a reliable method was established which can be used in clinical and pharmacological trials.
Diabetes Mellitus Alters the Effect of Peptide and Protein Ligands on Membrane Fluidity of Blood Platelets
Authors: Watala Cezary , Gwoździński Krzysztof , Pluskota Elżbieta , Pietrucha Tadeusz , Walkowiak Bogdan , trojanowski Zygmunt , Cierniewski Czesław S
Published: July 2018

The increased nonenzymatic glycosylation of platelet membrane proteins has been suggested to underlie platelet hypersensitivity in diabetes and the relationship of this to the reduced membrane lipid fluidity has been reported. As the modulation in membrane fluidity may determine the degree of accessibility of membrane receptors, the consequent alterations in membrane lipid-protein interactions in diabetes mellitus may also underlie the differentiated effects of various thrombotic and fibrinolytic agents on platelet membrane lipid bilayer.

In the present study we employed electron paramagnetic resonance and fluorescence spectroscopy to explore the ligand-induced platelet membrane fluidity changes in diabetic state, i.e. under conditions when the membrane architecture is considerably altered.

The yield of the excimer formation of pyrenemaleimide (PM), which depends directly upon the collisional rate and distances between molecules, was elevated in diabetic platelet membranes, thus pointing to the occurrence of some constraints in the structure/conformation of platelet membrane proteins in diabetes mellitus. Such an immobilization of PM was accompanied by the significant elevation in membrane protein gly-cation in diabetic platelets. The effects of various interacting ligands on platelet membrane fluidity were significantly lower in diabetic platelets, and the differences were much more distinct at the lower depths of a lipid bilayer. Nevertheless, the alterations in membrane lipid fluidity observed upon the interaction of a given ligand occurred with an approximately equal frequency in control and diabetic platelets. Moreover, the probability that these alterations were less profound in diabetic platelets was the same for all types of ligands studied. In diabetic patients the interaction of RGDS and tissue-type plasminogen activator with platelet membranes resulted in much smaller reductions of the h+1/h0 parameters in 5-DOXYL-Ste acid-labelled platelets, thus indicating a lesser rigidization of membrane lipid bilayer in diabetes. Likewise, the fluidizing effect of both fibrinogen itself and fibrinogen-derived peptides containing γ-chain carboxy-terminal sequence H-12-V was less pronounced in diabetic platelet membranes.
Detection of a Hypercoagulable State in Nonvalvular Atrial Fibrillation and the Effect of Anticoagulant Therapy
Authors: Mitusch Rolf , Slemens Hans J , Garbe Michael , Wagner Thomas , Sheikhzadeh Abdolhamid , Diederich Karl W
Published: July 2018

The purpose of the study was to evaluate alterations of the hemostatic system and the effect of anticoagulant therapy in nonvalvular atrial fibrillation. A set of molecular hematologic markers was measured prospectively in 69 patients with atrial fibrillation and 28 age-matched patients in sinus rhythm. Significantly elevated levels of thrombin-antithrombin III complex (8.5 ± 1.6 vs. 2.5 ± 0.3 αg/1; p <0.001), fibrin monomers (27.1 ± 3.2 vs. 13.4 ± 3.7 nM; p <0.001), D-dimers (788 ± 76 vs. 405 ± 46 αg/l; p <0.005), and tissue-type plasminogen activator (9.6 ± 0.5 vs. 7.2 ± 0.5 αg/l; p <0.05) were observed in patients with atrial fibrillation compared to those in sinus rhythm. In a subgroup of patients in whom anticoagulant therapy with oral coumadin or standard intravenous heparin was established after the initial study, hemostatic activation decreased significantly. In conclusion, molecular hematologic markers indicate a hypercoagulable state in atrial fibrillation which may characterize a group of patients at elevated risk for thromboembolic disease.
Phenotyping and Genotyping of Coagulation Factor V Leiden
Authors: Engel H , Zwang L , van Vliet H H D M , Michiles J J , Stibbe J , Lindemans J
Published: July 2018

The currently used activated Protein C resistance test demonstrated to be of limited diagnostic value for the detection of the mutant Factor V Leiden. Moreover, this assay is not useful for patients under anticoagulant therapy. A modification of the APC resistance test, applying Factor V deficient plasma is described which demonstrates a specificity and sensitivity of 1.0. The superiority of the modified APC resistance test over the existing APC resistance test was verified by genotyping.

For that purpose, the Amplification Refractory Mutation System (ARMS) was applied to the detection of the G to A mutation at position 1691 in the gene encoding coagulation Factor V. The mutation at that position could be easily detected by using each of two allele-specific oligonucleotide primers concomitantly with one common primer in two separate polymerase chain reactions, thereby amplifying a fragment of 186 base-pairs of the Factor V gene.
Factor V Gene Mutation Is a Risk Factor for Cerebral Venous Thrombosis
Authors: Martinelli I , Landi G , Merati G , Cella R , Tosetto A , Mannucci P M
Published: July 2018

To evaluate the association between coagulation defects and cerebral venous thrombosis, a case-control study was conducted in 25 patients who had no autoimmune, neoplastic or infectious disease and 75 healthy individuals. There were no patients with deficiency of protein C or protein S. Four had resistance to activated protein C (APC) and one had APC resistance associated with antithrombin deficiency. APC resistance was investigated by DNA analysis, and diagnosed by the presence of a point mutation in the factor V gene, which predicts replacement of Arg506 with Gin at one of the two APC cleavage sites in activated factor V. The prevalence of APC resistance was 20% in patients and 2.7% in controls. This difference was statistically significant (p = 0.01) and the odds ratio was 9.1. A circumstantial factor predisposing to cerebral venous thrombosis (such as oral contraceptive intake, pregnancy, puerperium, trauma or prolonged immobilization) was reported in 72% of cases. In conclusion, APC resistance is the most frequent coagulation abnormality associated with cerebral venous thrombosis.
Characterization of a cDNA Encoding Murine Coagulation Factor VII
Authors: Idusogie Esohe , Rosen Elliot , Geng Jie-Ping , Carmeliet Peter , Collen Desirá , Castellino Francis J
Published: July 2018

The cDNA encoding murine coagulation factor VII (mfVII) was isolated and reconstructed from a Λ Zap cDNA library generated from murine liver mRNA. The cDNA contains 1903 nucleotides spanning 15 bases upstream of the 5’-translation initiation codon, an open reading frame of 1338 nucleotides, 550 nucleotides downstream of the first stop codon and a 3’ poly(A) tail. The translation product is composed of a 41-amino acid signal/propeptide region followed by a 405-residue mature protein. The latter is highly homologous to that of human, rabbit, bovine, Rhesus monkey, and canine fVII. All protein domains of hfVII are strictly conserved in mfVII.
Comparison of Two Warfarin Regimens in the Prevention of Venous Thrombosis following Total Knee Replacement
Authors: Francis Charles W , Pellegrini Vincent D , Leibert Kristin M , Totterman Saara , Azodo Michael V , Harris Carl M , Cox Christopher , Marder Victor J
Published: July 2018

A prospective, randomized trial was conducted to compare the effectiveness and safety of warfarin given in two regimens in prevention of venous thrombosis after total knee replacement. Adult patients scheduled for primary or revision total knee replacement were randomly assigned to receive either a “two-step” warfarin regimen beginning 10-14 days pre-operatively or, alternatively, to begin warfarin the night before surgery. Post-operatively, the dose was adjusted in both groups to achieve a target International Normalized Ratio (INR) of 2.2 and prophylaxis was continued until venography on post-operative days five through nine. Bleeding was assessed by surgical blood loss, transfusion requirements, changes in hematocrit, and clinically identified bleeding complications. The occurrence of deep vein thrombosis was nearly the same in the two treatment groups, 39% in patients randomized to the two-step regimen as compared to 38% in those beginning the night before surgery. The occurrence of proximal vein thrombosis was also similar, 5% versus 7% (p = NS). Patients in the two-step group received 1.33 ± 1.26 transfusions compared to 0.95 ± 1.22 in the night before group (p <0.05) and also had a lower nadir post-operative hematocrit of 26.7 ± 3.1 as compared to 28.5 ± 3.2 (p <0.0001). Major bleeding complications were associated with excessively prolonged INRs and occurred in five patients in the two-step group and two in the night before group. Patients in both groups who developed thrombosis had a significantly lower INR on post-operative days two and three compared to those without thrombosis. We conclude that a prophylactic warfarin regimen for prevention of deep vein thrombosis after total knee replacement beginning the night before surgery is more convenient and may be associated with less bleeding than a regimen beginning warfarin 10-14 days pre-operatively. Careful control of anticoagulant intensity is needed to achieve maximum effectiveness and avoidance of bleeding complications.
Inhibitory Effect of a Synthetic Prostacyclin Analogue, Beraprost, on Urokinase-type Plasminogen Activator Expression in RC-K8 Human Lymphoma Cells
Authors: Nomura Naoki , Niiya Kenji , Shinbo Masahiro , Ozawa Tetsuo , Hayakawa Yumiko , Higashiyama Ko-ichi , Fujimaki Masao , Sakuragawa Nobuo
Published: July 2018

Plasminogen activation by urokinase-type plasminogen activator (uPA)is implicated in tumor invasion and metastasis by the breakdown of extracellular matrix. We have recently demonstrated the inhibitory effect of cAMP on uPA gene transcription in RC-K8 human lymphoma cells (Biochim Biophys Acta 1268: 293-9, 1995). Prostacyclin produced by endothelial cells is shown to increase cellular cAMP levels by activating adenylate cyclase. We, therefore, examined the effect of a stable analogue of prostacyclin, Beraprost, on uPA production in RC-K8 cells. uPA activity gradually increased in the conditioned medium with time. Beraprost (0.1 nM-1.0 μM) inhibited uPA accumulation in a dose-dependent manner without affecting cell viability. Fibrin-zymography demonstrated that high and low molecular forms of uPA were present in the conditioned medium and that after Beraprost-treat-ment all forms of uPA decreased and no PA/PA inhibitor complex was present. Northern blot analysis revealed that after exposure to Bera-prost, uPA mRNA levels increased transiently and then rapidly decreased to below control levels. Treatment with Beraprost resulted in a rapid activation of cellular cyclic AMP-dependent protein kinase (PKA). Beraprost completely negated uPA gene expression induced by phorbol myristate acetate, an activator of protein kinase C (PKC). These results suggest that Beraprost inhibits uPA production by suppressing uPA gene expression through the PKA pathway and that PKA-mediated signals are dominant in uPA gene expression as compared to those medicated by PKC. This inhibition of uPA expression by a prostacyclin analogue may be an important fact to explain the mechanism of anti-metastatic effects of prostacyclin.
Hemostatic Disorder of Uremia: The Platelet Defect, Main Determinant of the Prolonged Bleeding Time, Is Correlated with Indices of Activation of Coagulation and Fibrinolysis
Authors: Mezzano Diego , Tagle Rodrigo , Panes Olga , Pérez Marcos , Downey Patricio , Muñoz Blanca , Aranda Eduardo , Bajra Pliar , Thambo Sergio , González Fernando , Mezzano Sergio , Pereira Jaime
Published: July 2018

Several parameters of primary hemostasis and markers of activation of coagulation and fibrinolysis were measured in 48 patients with severe (creatinine clearance <20 ml/min) chronic renal failure (CRF) without dialysis and diseases or drugs affecting hemostasis. Bleeding time (BT) was prolonged in 25/48 patients, and was correlated with age of patients, severity of renal failure, hematocrit, impairment in platelet aggregation-secretion and decrease in platelet ATP content. Defects in von Willebrand factor played no role in the prolongation of the BT. Multivariate analysis showed that only platelet dysfunction and severity of renal disease were independent predictors of the BT in uremia. The platelet functional disorder was significantly correlated with a reduction in platelet ATP and ADP.

High levels of plasma thrombin-antithrombin complexes (TAT), prothrombin fragment F1+2, fibrinogen and factor VIIc were observed in patients with CRF, as described in prethrombotic states. Plasmin-antiplasmin complexes (PAP), fibrinogen and fibrin degradation products (FgDP, FnDP) were significantly increased, and the activity of plasminogen activator inhibitor (PAI-1) was slightly reduced, denoting an activation of fibrinolysis.

A negative correlation was found between platelet levels of ATP and ADP with plasma TAT, F1+2 and PAP. Furthermore, plasma PAI-1 activity was negatively correlated with the BT and was lower in patients with prolonged BT as compared with controls and patients with normal BT. These links between primary hemostasis and activation of coagulation and fibrinolysis suggest that increased intravascular generation of thrombin and/or plasmin is an important mediator of the defects in primary hemostasis, prolongation of the BT and, probably, bleeding in CRF.
A Method For Rapid Visualization Of The Size- Distribution Of Factor VIII-Related Protein In Plasma
Authors: Perret B A , Felix R , Furlan M , Beck E A
Published: July 2018

Factor VUI-related protein circulates in normal human plasma as a series of multimeric forms with apparent molecular weights ranging from 1 to 20×106. So far, combined electrophoretic and immunologic methods permitted demonstration of variable concentration and size- distribution of factor VIII-related protein in von Willebrand’s disease as compared to normal. We now have devised a one-step method for determining the size pattern of this plasma protein. Fresh plasma, containing 1% SDS and 0.8M urea, was layered on top of 2.5% polyacrylamide gels with 2.75% by weight of methylene bisacrylamide/acrylamide. Following extended electrophoresis in 0.2% SDS-0.1M Tris/HCl (pH 7.4), the gels were soaked in 5% formaldehyde and then extensively washed with 10% ethanol. Proteins were visualized employing an ultrasensitive ammoniacal silver stain. This staining revealed a multimeric protein pattern in the upper part of the gel the distribution of which was recorded by densitometry. The protein was identified as factor VIII by two-dimensional immunoelectrophoresis. The method was reproducible and allowed densitometric evaluation within 24 hr.
Antibodies Against Platelet Membrane Glycoproteins: Effect On Ristocetin-Induced Platelet Aggregation
Authors: Ali-Briggs E F , Jenkins C S P , Clemetson K J
Published: July 2018

Some membrane glycoproteins (GPs) have been isolated by lectin-affinity chromatography and antibodies towards them have been raised. Platelets that have lost glycocalicin no longer respond to ristocetin-human VIII:WF, bovine VIIIR:WF, or to anti-glycocalicin or anti-GPs la and lb antibodies but are still agglutinated by anti-GPs lib and Ilia antibodies. Anti-GPs la and lb and anti-glycocalicin antibodies, IgG and Fab' fragments inhibited ristocetin- human VIIIR:WF- and bovine VIIIR:WF-induced aggregation of fixed, washed platelets and of platelets in plasma while anti-GPs Hb and Ilia antibodies were without effect.

Crossed immunoelectrophorectic studies showed that glycocalicin was present on whole platelets in only trace amounts; anti-glycocalicin antibodies, however, recognized a slower migrating component. Platelets incubated in an EDTA-free medium no longer respond to ristocetin-human VIIIRrWF. Membranes isolated from such platelets contained glycocalicin which cross-reacted with a remnant of the slower migrating component. Anti-GPs la and lb antibodies gave more complex patterns but it was possible to identify the slower moving component recognized by the anti-glycocalicin antibodies.

These results show that glycocalicin is not normally found as such on whole platelets but is present as a precursor which is most likely GP lb. On degradation of this precursor, glycocalicin is released from the membrane and VIIIRrWF-receptor activity is lost.
Injury Of Cultured Endothelial Cells By Thrombin-Stimulated Platelets
Authors: Jørgensen L , Grøthe A G , Larsen T , Kinlough-Rathbone R L , Mustard J F
Published: July 2018

Platelets stimulated in vivo may cause endothelial injury. The question is whether a similar effect of platelet stimulation may be shown in vitro.

Endothelial cells were cultured from human umbilical veins in Medium 199 with 20 per cent heat-inactivated serum. Semiconfluent cultures, 100-400.000 cells per dish, were labelled with Na2Cr51O4. Twenty-four hours later human platelet suspension (final cone. 200.000 per mm3) and thrombin (final cone. 4 U/ml) were added to the medium and the culture dish shaken for 15 min. The percentage of cells detached from the culture dish and the percentage of Cr51 released from the endothelial cells into the ambient fluid during the shaking were determined and used as parameters of cell injury. Increased percentages of loosened cells and Cr51 into the ambient fluid were observed with platelet suspension and thrombin compared to controls without platelet suspension and/or thrombin. Platelet-free supernatant after reaction of thrombin with platelet suspension had a similar effect. Thrombin alone caused a moderat release of Cr51, but no increased loosening of cells. Both parameters increased with increasing dose of thrombin when the platelet count was kept constant. The percentage of loosened cells increased with increasing platelet number when the dose of thrombin was constant. A significant release of Cr51 occurred only at high concentrations of platelets. Scanning and transmission electron microscopy of endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injured cells in association with platelets. The endothelial cells were bulging, intercellular junctions opened up, the plasma membrane on the luminal side showed villi and breaks and the cytoplasm had increased electron density. Thus, platelets stimulated by thrombin may cause injury of endothelial cells also in vitro.
Effects Of Aspirin And Sulfinpyrazone On Platelets, Vascular Cells And Their Interactions
Authors: Buchanan M R , Vazquez M J , Gimbrone M A
Published: July 2018

Sulfinpyrazone (SUL) and aspirin (ASA) are potentially useful antithrombotic drugs. Both drugs are thought to exert this effect by inhibiting the platelet enzyme, cyclooxygenase (C-0), thus preventing thromboxane A2 synthesis. Recent data, however, suggest that these drugs also may affect vessel wall cells. To study this further, we examined the effects of SUL and ASA on i) the adhesion of 3H-adenine-labelled washed human platelets to cultured bovine endothelial (EC) and smooth muscle cells (SMC), ii) EC and SMC DNA synthesis (3H-thymidine incorporation) and iii) cell growth. Pretreatment of platelets with 100μM ASA or 250μM SUL (concentrations sufficient to inhibit C-0), did not affect platelet adhesion to untreated EC or SMC. However, adhesion of untreated, ASA- and SUL-platelets was increased 25,28 and 44% resp. when EC were pretreated with 650μM SUL for 24 hr. In contrast, adhesion of ASA-platelets to EC pretreated with lOOμM ASA (sufficient to inhibit prostacyclin), was unaffected. Platelet adhesion to SMC pretreated with 650μM SUL for 24 hr was decreased when platelets also were pretreated with ASA (20%, p<0.05) or SUL (27%, pc 0.02). Pretreatment of SMC with SUL for only 2 hr had no effect. DNA synthesis in EC and SMC treated with 62.5 and 250μM SUL for 24 hr, was inhibited >35% and >95% resp. Preliminary data suggest that this inhibitory effect may last longer in SMC. To study the effect of SUL on cell growth, EC and SMC were plated at 2 × 104 cells/ cm2 and fed with culture medium containing 0, 62.5 or 625uM SUL on day 0, 1, 3 and 4.5. EC growth rate and final density were unaffected over 7 days. SMC growth rate also was unaffected, but the final density of SMC treated with 650μM SUL was 31 μ 2% less than untreated SMC at 7 days (p<0.01). These data indicate that SUL has direct effects on EC and SMC that may influence i) platelet-vessel wall interactions and ii) vascular cell proliferation.
Reconstitution Of PF1 In Platelets From Factor V-Deficient Donors As A Function Of Available PF3
Authors: Bode A P , Sandberg H , Dombrose F A , Lentz B R
Published: July 2018

Reconstitution of membrane-associated Factor V-like activity (PF1) in human platelets isolated from severe F.V-deficient donors was assessed following incubations in citrated normal platelet free plasma. Coagulant activities were measured using: a one-stage prothrombin time, the Stypven time and a modified KAPTT in which the kaolin particles were removed from the plasma prior to recalcification. The supernatant from 3x frozen-and-thawed lysed normal platelets was used as a standard for 100% PF1 and 100% PF3. Normal platelets gel filtered or centrifuge-washed in the presence of adenosine, theophylline and PGEj had <1% available PFI and PF3, whereas platelets from severe F.V-deficient donors isolated by the same procedure had no PF1 and the same amount of PF3. The supernatant from 3x frozen-and-thawed lysed F.V-deficient platelets also had no PF1 but had 100% total PF3. When gel filtered, PFl-deficient platelets were incubated in normal plasma, they acquired about 1% PF1 which did not change following freezing-and-thawing. By contrast, PFI-deficent platelets washed in the absence of these inhibitors had 15-20 times more available PF3 and acquired about 15-20 times more total PFI after incubation in normal plasma. When either the crude frozen-and-thawed lysed membrane supernatant from these PFl-deficient platelets (100% available PF3) or a partially purified membrane-rich fraction from this supernatant was incubated with normal plasma, then 100% reconstitution of PFI was achieved. PF1 was also reconstituted in a purified system using all-or-none binding when the PF3-containing lipid-protein particles secreted by platelets, upon collagen induced aggregation, were incubated with purified human F.Va. Following incubation, these particles had the same amount of PFI as the PF3-containing particles from normal platelets. It is apparent that in human platelets a correlation exists between the amount of available PF3 and the capacity to reconstitute PFI.
Direct Assay For Factor XII In Plasma
Authors: Zuffi T , Jordan R
Published: July 2018

A chromogenic substrate assay for FXII in plasma samples has been developed. The method involves 0°C activation of the contact factors with dextran sulfate, and a direct selective measurement of the FXIIa activity generated by hydrolysis of the synthetic substrate S2302 in the presence of the kallikrein inhibitor, soybean trypsin inhibitor. Using these procedures, the kinetics of both FXII and pre- kallikrein activation could be studied and it was found that the relative rates of activation of these two zymogens did not necessarily coincide.

When this dextran sulfate treatment of FXII or HMWK-deficient plasmas was tried, insignificant kallikrein or FXIIa activity was generated, demonstrating that contact activation was not occurring. These studies have shown that a dextran sulfate activation can be used as an assay procedure for FXII in plasma and can distinguish normal from certain deficient plasmas.
Protection Of Endotoxin Induced Disseminated Intravascular Coagulation In Rats By Gabexate Mesilate
Authors: Yoshikawa T , Furukawa Y , Murakami M , Takemura S , Kondo M
Published: July 2018

Gabexate mesilate[ methane sulfonic acid salt of ethyl-p- (6-guanidino hexanoyloxy) benzoate : FOY ] has recently been developed in Japan, and has been known to have potent inhibitory effects on trypsin, kallikrein, plasmin, thrombin and C1-esterase. Advantage of clinical use of this agent is that FOY has smaller molecular weight than aproti- nin so that production of antibody against FOY is hardly observed. In the present investigation, effect of FOY on disseminated intravascular coagulation (DIC) was examined using experimental animal models, in comparison with that of heparin.

Female rats of Wistar strain (8-weeks) were infused with l00mg/kg of bacterial endotoxin (Lipopolysaccharide B; E. coli; 055, Difco) continuously for 4 hours through femoral vein. Blood samples were serially taken from abdominal artery using catheter and examined for plasma fibrinogen, FDP, platelet counts, prothrombin time and partial thromboplastin time. After the experiment, kidneys were removed to examine the deposition of fibrin to glomeruli.

Different concentrations of FOY were intraperitoneally injected to the rats prior to the infusion of endotoxin, and it was found that the administration of 10 mg/kg of FOY showed the most potent inhibitory effect on the development of DIC, either hematologically or histologically. In comparison, heparin showed a strong inhibitory effect on DIC over a dosage of 5 U/kg.

It is concluded that, although inhibitory effect of FOY was less significant than heparin, FOY might be valuable agent for the treatment of DIC especially when heparin is difficult to use in such cases as severe hemorrhagic tendency.
Heparin Chromatography
Authors: Losito R , Gattiker H , Bilodeau G , Longpré B
Published: July 2018

Commercial heparin is not a homogeneous substance. The isolation of the active component still appears to be a problem and usually requires several different procedures. In order to see if it would be possible to simplify the purification, we studied and compared the chromatography of porcine heparin by a systematic approach using various types of adsorbants, gels and programmed elution techniques employing a Beckman Spectrochrom 130 chromatographic analyzer. A total of 20 combinations were attempted with 200 mg of heparin (169 u/mg) being chromatographed in each case. The fractions of each peak were tested by several methods and included the activated partial thromboplastin time, recalcification time, thrombin time, anti-factor Xa, chromogenic, USP assay, agarose gel electrophoresis and the measurement of metachromatic activity. It was found that dextran gel with an average dry diameter of 75-100 um produced the best chromatograms whose peaks were well resolved. The medium molecular weight fraction of the second peak contained half of the applied heparin. This peak contained all the anticoagulant activity besides reacting very strongly for metachromatic activity. The smaller and larger molecular fractions found in the other three peaks gave very weak metachromatic activity and were devoid of anticoagulant activity. These results have important implications in the preparation and clinical use of heparin.
The Effect Of Plasmin On Factor XIII (Fibrin Stabilizing Factor)
Authors: Rider D M , McDonagh J M
Published: July 2018

The action of plasmin on several blood clotting factors has been studied; however, controversy exists concerning the effect of plasmin on factor XIII. Factor XIII was purified from plasma and platelets and then exposed to plasmin for up to 6 hours. Plasmin to factor XIII ratios ranged from 0.03-0.1 casein units plasmin per mg factor XIII. These plasmin levels exhibited strong proteolytic activities against B-casein and purified human fibrinogen Following incubation of factor XIII (activated and unactivated) with plasmin the mixtures were electrophoresed on 7% SDS-polyacrylamide gels. The factor XIII preparations were assayed for 14C-putrescine incorporating activity before and after exposure to plasmin. Platelet factor XIII was,labeled With 125Iodine and lableled a subunit (activated and unactivated) was exposed to plalmin for up to 2 hours. These mixtures were electrophoresed on 12.5% Urea-SDS Polyacrylamide gels and a radioactivity profile was determined for each gel.

Following extensive exposure to Plasmin the relative molecular weights of the factor XIII subunits (a, a* and b)remained constant and almost all (90-100%) of the 14C-put-rescine incorporating activity was recovered. The radio-activity profiles of the gels of 125I-labeled platelet factor XIII were identical before and after incubation with plasmin. Plasmin did not activate factor XIII in the assay system nor did factor XIII inactivate plasmin by crosslinking it. These experiments indicate that plasmin does not activate or degrade factor XIII and that the b subunit of plasma factor XIII plays no role in protecting the a subunit from the action of plasmin.
Blood Fluidity In Chronic Occlusive Arterial Disease-Effects Of Pentoxifylline
Authors: Angekort B
Published: July 2018

Function of microcirculation depends largely on the fluidity of blood. The study aimes with the influence of risk factors and coagulation phenomena on blood flow in capillaries typical for chronic occlusive arterial disease. (COAD) Investigations of blood fluidity base on passage rates of EDTA-anticoagulated blood through a 5,um micropore filter system in simulation of capillary conditiones. The flow rates in COAD show marked decrease of erythrocyte flow compared to healty subjects (1,8 ± 0,2 and 2,9 ± 0,3 ml/min). With heavy smoking, hypercholesteroT- and hypertriglyceridemia the flow rates in otherwise healty persons accord with those in COAD. After normalisation of lipids flow rates are normalised. Isolated coagulation phenomena typical for hypercoagulability in COAD result in significantly reduced flow rates whereby a decreased fibrinogen level has no influence on blood fluidity. The data point to the necessity of therapeutic elimination of aforementioned risk factors as well as improvement of erythrocyte fluidity and removal of hypercoagulabi1ity in COAD. Therapeutic oral administration of pentoxifylline in COAD disclosed improvement of erythrocyte fluidity (flow rates before/after: 1,7 ± 0,3 to 2,5 ± 0,3 ml/min), decrease of platelet hyperreagibi1ity and plasmatic hypercoagulation as well as an increase of fibrinolytic activity of the blood.
Discrimination Between Prostglandin-Dependent And-Independent Functions Of Essential Fatty Acids In Arterial Throm- Bogenesis
Authors: Houtsmuller U M T , Hornstra G , Haddeman E
Published: July 2018

Arterial thrombus formation is reduced in essential fatty acid (EFA) deficiency. This goes together with an enhanced thrombin induced aggregation of platelets in vitro,whereas collagen-induced aggregation is definitely suppressed. A small amount of linoleic acid (18:2 (n-6)) is able to cure EFA-deficiency and to normalize arterial thrombogenesis. This latter effect may be due to either the structural function of this EFA or to its function as the ultimate dietary precursor of prostaglandins (PG). Columbinic acid, a stereo-isomer of γ-linolenic acid (18:3 (n-6)) was recently shown to possess all the structural functions of EFA, but not the PG-dependent ones. This fatty acid therefore presents a suitable tool to investigate the PG-dependence of arterial thrombogenesis and its underlying processes. We therefore compared the effect of small amounts of linoleic and columbinic acid (both as methylesters) on the water vapour release in vivo (which is a sensitive parameter for a non-PG dependent function of polyenoic fatty acids), arterial thrombosis tendency (time needed for the thrombotic obstruction of an aorta prosthesis) and platelet aggregation in vitro (aggregometry) induced by collagen and thrombin. In contrast to linoleic acid, columbinic acid did not normalize arterial thrombosis tendency and collagen induced platelet aggregation. Columbinic acid was equally effective as linoleic acid in the normalization of the water vapour release in vivo and of the thrombin-induced aggregation. We conclude that arterial thrombus formation and collagen- induced aggregation greatly depend on prostanoid formation, whereas thrombin-induced aggregation does not. The structural role of polyenoic fatty acids in thrombin-induced aggregation may provide a tool in the elucidation of factors determining the thrombin-sensitivity of blood platelets.