bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat-Induced Fragmentation Of Human Plasma Fibronectin
Authors: Miekka Shirley I
Published: July 2018

Human plasma fibronectin, purified by affinity chromatography on gelatin-Sepharose, undergoes fragmentation during heat treatment at 100°C prior to reduced SDS-PAGE. The electrophoretic patterns showed a progressive decrease in the intensity of the 230 kd doublet characteristic of reduced fibronectin, which correlated with the appearance of increasing amounts of smaller components having molecular weights ranging from 30-200 kd. Fragmentation was temperature dependent, being undetectable after 2 hr at either 22° or 60°C, but clearly evident after 30 min at 70°C and above. After 2 hr at 100°C the intact 230 kd monomer was no longer visible. The effect was also pH dependent; fibronectin samples heated for 20 min at 100°C in SDS solutions buffered at pH 2.5, 4, 7 and 10 exhibited maximum degradation at pH 4. When heated in the absence of SDS, the protein was partially aggregated to polymers which did not enter the gel, but fragments were still observed. Equivalent results were obtained whether mercaptoethanol was added before or after the heat incubation step.

The fragmentation does not appear to be due to proteolysis. Pretreatment with various combinations of inhibitors of serine-, thiol- or metallo-proteases did not detectably diminish the effect. Sterile filtration through microporous membranes (0.22 or 0.45 uM) to remove possible cellular contaminants did not improve the stability. Furthermore, when mixtures of fibronectin and fibrinogen were heated (20 min, 100°C) the fibrinogen chains were not detectably altered while fibronectin was fragmented as usual.

These observations suggest that heat-induced fragmentation of fibronectin reflects an intrinsic instability of the molecule at high temperatures, perhaps analogous to that observed with α2-macroglobulin and complement components.
Fibronectin In Commercial Coagulation Factor Concentrates And Response Of Fibronectin Plasma Levels To Infusions
Authors: Gomperts Edward D , Izadi P , Berg D
Published: July 2018

Fibronectin (FN) was assayed via a commercial mono-specific antiserum (Cappell Labs Inc.) using a Laurel immuno- electrophoretic assay with a pool of 10 normal plasmas as control. This control pool was arbitrarily given the FN concentration of 100% containing 300 μg FN/ml plasma. FN was measured in various lots of different commercial factor concentrates and cryoprecipitate. The concentration of FN varied widely among 3 different commercially available FVIII concentrates. One product showed a mean level of 2,376% (6 lots), a second 415% (4 lots) and a third 903% (2 lots). The average FN level of 3 cryoprecipitate preparations was 576%. FN was not present in 3 lots of one commercial FIX concentrate. FN was assayed in the plasma of 12 patients with hemophilia before and immediately after factor concentrate/cryoprecipitate infusion for therapeutic purposes. The response of the plasma FN level to the infusion was assessed by determining the total FN infused (mg), the observed rise in plasma (%) and the absolute FN amount producing a 1% rise in plasma level. The quantity of FN infused (a function of volume of infusate and concentration of FN) varied widely from 1.19 mg to 17.0 mg total. The observed rise depended inter alia on absolute FN infused and body weight. This ranged between 0 and 139%. Cryoprecipitate produced the greatest rise of plasma FN level per unit weight of FN infused. The commercial concentrates showed a wide spectrum of plasma FN responses in most cases requiring greater absolute FN infusion doses than cryoprecipitate for each 1% of increase in plasma level. These results indicate that FN is a major contaminant of some commercial Factor VIII concentrates. It is possible that FN is denatured by the concentration process resulting in very rapid removal of infused FN from the circulation.
The Source Of Thromboxane Formation Associated With Complement-Mediated Pulmonary Leukostasis In Sheep
Authors: McDonald J W D , Ali M , Cooper J D , Townsend E R
Published: July 2018

The infusion of plasma containing Zymosan-activated complement (ZAC) into sheep produces leukopenia with pulmonary leukostasis and transient pulmonary arterial hypertension (PAH). Previous work has related PAH to elevations of plasma thromboxane B2 (TXB2) rather than to mechanical obstruction by sequestered leukocytes (WBC). We have investigated the source of the TXB2 formation in this model. Incubation of platelet-poor WBC preparations with arachi- donate resulted in negligible TXB2 formation. WBC-poor platelet preparations on the other hand formed significant amounts of TXB2 (approximately 6-18 ng/108 platelets). Incubation of whole sheep blood or plasma with ZAC failed to generate significant amounts of TXB2. Thus, WBC agglutination in vitro did not induce platelet TXB2 formation.

Pretreatment of sheep with aspirin (ASA)(10 mg/kg IV) completely blocked TXB2 formation and PAH in response to infusion of plasma containing ZAC. The infusion of 10-50% nonnal platelets into sheep 4 hours after ASA pretreatment failed to restore TXB2 formation and pulmonary vascular response to subsequent challenge with ZAC. TXB2 formation during blood clotting ex vivo was restored by the platelet infusions. These experiments make it appear unlikely that platelets are the source of the TXB2. It is possible that the transfused platelets respond to thrombin but are unable to interact with sequestered leukocytes. Sheep lung and pulmonary artery were incubated in vitro with arachidonate. Lung formed 630 ng TXB2 and 39 ng 6-keto-PGF1α/g of wet tissue. Pulmonary artery formed 9 ng TXB2 and 180 ng 6-keto-PGF1α/g of wet tissue. The relative proportions of TXB2 and 6-keto-PGF1α formed by lung parenchyma but not pulmonary artery resemble the proportions observed in previous in vivo experiments with ZAC. It appears that lung tissue is the most likely source of TXB2 formation causing PAH in response to ZAC-mediated pulmonary leukostasis.
Genetic Predisposition To The Expression Of Lymphoid Procoagulant Activity (PCA) In Response To Viral Stimulation
Authors: Levy G A , Leibowitz J L , Edgington T S
Published: July 2018

A predisposing foctor in the induction of viral induced disease may be the genetic make-up of the host. Leukocytes from a variety of species uniformly generate increased amounts of procoagulant molecules in response to a variety of stimuli including antigen:antibody complexes, lipopolysaccharides (LPS), complement fragments C5a and lectins. Although the amount of increased PCA varies with the concentration and duration of contact with the stimulus, all individuals respond to equivalently these stimuli. The coronavirus family is a group of RNA viruses and includes the murine hepatitis virus (MHV) which causes strain dependent forms of disease in mice. BaIb/C and DBA/2 mice develop acute necrotizing hepatitis, C3H mice develop a mild chronic disease and the A strain develops no evidence of hepatitis. Peripheral blood mononuclear cells from BaIb/C and DBA/2 mice respond both in vitro and in vivo with a marked increase in procoagulant activity (PCA), with 106 PFU of MHV-3 invoking an 80-fold increase in PCA and 10 PFU a 10 fold increase in PCA. PBM from C3H strain mice respond to MHV with a 6.8-fold increase of PCA at 106 PFU of virus. In constrast, PBM from A strain mice do not respond to 10-106 PFU or virus. The PCA was a product of monocytes as demonstrated by direct cytologic assay, however induction of PCA required the presence of lymphocytes. Two types of PCA were found, an early increase of a novel cellular prothrombinase and later increase in thromboplastin activity. Thus, the expression of MHV induced PCA appears to be genetically restricted and also correlates with susceptibility to viral induction of disease. The existence of comparable genetic restrictions in man might relate the variable incidence of thrombotic diseases. Supported by NIH grants HL- 16411 and CA-28166.
Dissociation Of Aprotinin-Human Plasmin Complex By Fibrin
Authors: Vairel E G , Thely M , Brouty-Boye H
Published: July 2018

Aprotinin inhibits plasmin by forming an inactive complex which has a binding force of 3.108.

When the complex, in a 0.15M buffer solution at pH 7.4, is percolated through a column of human fibrin balanced with the same buffer, all of the aprotinin appears promptly in the eluent and the fibrin column dissolves progressively from the top. If the buffer is replaced with a 0.15M epsilon amino-caproic acid solution as soon as lysis begins, the fibrinolysis is stepped and plasmin is eluted.

If citrated human plasmin is used in a 1:1 solution with buffer, the same events take place but over a longer period of time ; the aprotinin appears slowly in the eluent and fibrinolysis begins later.

It thus appears that fibrin is able to dissociate the aprotinin-plasmin complex, probably by forming a shortlived ternary complex. The complex binds to fibrin even in a plasma medium, and fibrinolysis occurs even with an excess oxsf plasmic inhibitors in the medium.

Fibrinogen does not possess this property of fibrin. The complex can be detected in the circulating blood several hours after intravenous administration to rabbits.

This property of fibrin suggests a possible use of the aprotinin-plasmin complex as specific fibrinolytic treatment.
The In Vitro Effect Of Ticlopidine On Fibrinogen And Factor VIII Binding To Human Platelets
Authors: Lee H , Paton R C , Ruan C
Published: July 2018

Ticlopidine is an anti-aggregating drug whose mode of action is not yet fully understood. Its multiple effects on platelet function include prolonged bleeding time, reduction in primary and secondary waves of ADP-induced aggregation and inhibition of collagen and thrombin- induced aggregation. We have studied the in vitro effects of ticlopidine on fibrinogen binding induced by ADP and adrenaline as well as Factor VIII/vWF binding induced by ristocetin.

125I fibrinogen binding was measured in suspensions of freshly-washed normal platelets stimulated by 10 μM ADP or 10 μM adrenaline. The binding of 125I-Factor VIII/vWF in the presence of 1 mg/ml ristocetin was measured in both washed and paraformaldehyde-fixed platelets. Ticlopidine at final concentrations of 200, 100, 50 and 25 μM inhibited both ADP and adrenaline-induced fibrinogen binding in a dose-dependent manner. The mean % inhibition of ADP-induced fibrinogen binding was 82, 73, 42 and 32 respectively. The mean % inhibition of adrenaline-induced fibrinogen binding was 86, 82, 60 and 35 respectively. In contrast, the Factor VIII/vWF binding was unaffected by ticlopidine at all concentrations except at 200 μM using fresh platelets where a slight inhibition (19 %) was observed.

These results suggest that ticlopidine either inhibits platelet activation and consequently fibrinogen binding, or inhibits the binding directly, presumably by having an effect on the specific configuration of the platelet membrane required for normal fibrinogen binding.
Piracetam: A New Platelet Suppressing Drug
Authors: Bick R L , Fareed J , Skondia V
Published: July 2018

Piracetam, 2-Oxy-1-pyrrolidine Acetamide is a cyclic derivative of gamma-aminobutyric acid and has recently undergone successful human trials as a platelet-suppressing agent. These investigations were designed to study the mechanism of action of Piracetam in suppressing platelet function in human subjects. Twenty-eight subjects were studied; these individuals ingested Piracetam at 9.6 gm/day in four divided doses. Template bleeding times, platelet adhesion (Bowie technique) and platelet aggregation to collagen, ristocetin, serotonin, ADP, epinephrine, and arachidonic acid was performed before and one week after beginning ingestion of this drug. All individuals demonstrated prolongation of the template bleeding time; the average prolongation was 6 min. over baseline bleeding times. Platelet adhesion became abnormal in the majority of individuals during ingestion of Piracetam, but the degree of abnormality was not consistent. However, consistent aggregation abnormalities were induced by Piracetam. All individuals showed absence of the second wave of epinephrine at 2.7 uM and 5.4 uM, all individuals demonstrated abnormal aggregation to low concentration ADP (2.0 uM) and most were abnormal to high concentration ADP (6.6 uM). In addition, all individuals demonstrated absence of arachidonic acid-induced aggregation after ingestion of Piracetam. No consistent abnormalities of collagen or ristocetin-induced aggregation were noted. The results of this study suggest that Piracetam has at least two sites of action in suppressing platelet function. One site of action appears to be inhibition of thromboxane systhesis at some, as yet undetermined, site in the platelet prostaglandin synthetic pathway, and the other mechanism appears to be inhibition of release by platelet dense bodies. It is suggested that the presence of at least two sites of action may make this non-toxic agent a highly useful antiplatelet drug.
Retraction Of Fibrin Clots By Cultured Endothelial Cells
Authors: Barbieri B , Balconi G , Dejana E , Donati M B
Published: July 2018

The interactions between endothelial cells (EC) and fibrin are still poorly understood. We approached this problem by studying the ability of cultured EC to induce in vitro the retraction of fibrin clots. EC were obtained from bovine aorta and human umbilical veins by collagenase treatment and grown in Eagle MEM. At the time of the test the cells were harvested from the flask by a short trypsin-EDTA treatment and resuspended in tyrode solution. The test system involved incubation of the cell suspension in a water-bath at 37°C in the presence of cell-free plasma which was clotted by thrombin. The course of retraction was followed by measuring the diameter of the clot with a microcaliper. Retraction values were expressed after calculation of percent activity by an appropriate formula. EC were found to induce the retraction of the fibrin clot to an extent which increased with the time (1-24 h) and with the number of cells in the system (l-4×l06/ml f.c.). Fibrin clot retractile (FCR) activity of EC could not be detected at 22°C or in presence of Na2-EDTA or using mechanically disrupted cells. Moreover, using batroxobin instead of thrombin as a clotting agent, no retraction occurred; FCR of EC thus showed many characteristics in common with platelet- and fibroblast- induced clot retraction.

FCR activity of bovine EC increased with the number of subcultures, being very low in cells harvested from primary cultures. In contrast, human EC had high activity in primary cultures. Similarly to fibroblasts, EC with higher density in culture showed lower FCR, suggesting that con-fluency inhibits the cell contractile capacity.

FCR could thus represent a simple in vitro test to further characterize the biology of EC and to evaluate their role in the development of fibrin thrombi.
Pathogenesis Of Clinical Events In Coronary Artery Disease. Clinical Observations
Authors: Maseri A
Published: July 2018

Studies on angina at rest showing that an increase of myocardial demand beyond a fixed supply is not the only or even the most frequent cause of acute myocardial ischemia open a new avenue in our understanding of ischemic heart disease. Functional factors that transiently interfere with regional coronary blood supply, such as coronary spasm, seem to play a major role in determining the clinical events associated with coronary atherosclerotic heart disease. These functional factors appear to be the major cause of angina at rest, nocturnal, post-prandial and cold-induced angina and for variable threshold of exertional angina.

Sudden death was also shown to be a possible consequence of coronary spasm which was shown to cause ventricular fibrillation and cardiac arrest during reversible ischemic episodes with or without anginal pain.

Myocardial infarction consistently occurs in the same vascular bed shown to undergo transient ischemic episodes before the onset of the final, irreversible one. The transient episodes were shown to be caused by coronary spasm. The irreversible episode could not be relieved by intracoronary nitrates. A platelet mural thrombus was found at post mortem at the site of demonstrated persistent spasm.

We suggest that coronary vasospasm and platelet aggregation may be responsible for the onset of acute myocardial infarction and, possibly, for the development of coronary thrombotic obstructions.

Thus, the clinical manifestations of ischemic heart disease may largely depend on the presence and severity of functional factors, transiently and acutely interfering with flow, along with a variable degree of coronary atherosclerosis. In turn, prognosis may be determined by the severity of acute functional factors and the extent to which the coronary circulation and the myocardium are already chronically jeopardized by organic lesions.
Lack of Circulating Prostacyclin (PGI2) In Guinea Pig And Rat
Authors: Fisher J M , Willis A L , Smith D L , Donegan D
Published: July 2018

Under light ether anesthesia, blood of male guinea pigs (Hartley strain, 330-410 g, Simonsen, Gilroy, CA) or rats (Sprague-Dawley, 230-575 g, Simonsen) was withdrawn into sodiun citrate (0.38% w/v). Platelet rich plasm (PRP) was then prepared by a rapid centrifugation procedure. Aggregation induced by ADP (1.6 μg/ml) was then examined in 0.15 or 0.25 ml aliquots of PRP at various time intervals from 5-60 min after blood withdrawal. A spontaneous time-dependent rise in aggregation response occurred that was similar to that observed when PGI2 (0.5-3ng) was added to PRP in vitro. In both species, intraperitoneal ackninistration of indanethacin (100 mg/kg, 1h previously) failed to interfere with the spontaneous rise in aggregation although vascular PGI2 formation was shown to be virtually abolished. Similar results were seen in essential fatty acid (EFA) deficient rats that had been chronically maintained on a fat free diet. In these animals, vascular PGI2 product ion was less than 15% that of controls. These results clearly indicate that any basal levels of PGI2 present in the arterial circulation are less than those (0.3-1ng/ml) necessary to appreciably inhibit aggregation. This conclusion coincides with that of Steer, et al. (Nature, 283, 194, 1980) who failed to detect biologically active amounts of PGI2 in hiimn venous blood and of several other groups who have failed to detect significant blood levels of the PGI2 breakdown product, 6-keto-PGF1α.
Intrinsic Fluorescence Changes And Exposure Of Sulfhydryl Groups On α2-Macro Globulin During Reaction With Serine Proteases
Authors: Straight D L , McKee P A
Published: July 2018

The reactions of serine proteases, methyl amine (CH3NH2) and denaturants with α2-macroglobulin (α2M) were studied by intrinsic fluorescence, SDS gel electrophoresis and sulfhy- dryl (SH) group titration. The cleavage of α2M by trypsin, thrombin and elastase, as followed on SDS gels after reduction, corresponded to an increase in protein fluorescence. Both proteolysis and fluorescence were maximal at two moles of each enzyme per mole of α2M. The second order rate constant for inhibition of thrombin’s fibrinogen clotting activity by α2M was the same as the rate constant for fluorescence change during reaction of α2M with thrombin. Amines (e.g., CH3NH2) that inactivate α2M caused an increase in protein fluorescence that correlated with the rate of loss of α2M’s ability to protect trypsin from soybean trypsin inhibitor. Prior treatment of α2M with proteases or amines abolished the ability of either to enhance fluorescence. We found that CH3NH2 also caused exposure of ∼4 SH groups per molecule of α2M when titrated with 2,2’dipyridyl disulfide or 5,5’dithiobis (2-nitrobenzoic acid). The rate of SH group exposure by CH3NH2 corresponded to the rate of protein fluorescence change induced by CH3NH2. The reaction of α2M with proteases gave similar results. Denaturation of α2M in 6 M guanidine. HCl (GnHCl) at neutral pH also exposed SH groups, but denaturation in 6 M GnHCl at acid pH, where the stoichiometric reaction of amines with α2M occurs very slowly, did not expose SH groups. This indicates that a conformational change is not solely responsible for this phenomenon. We conclude: 1) the stoichiometric reactions of proteases and amines with α2M coincide with a measurable increase in protein fluorescence; 2) two moles of trypsin, thrombin or elastase bind to one mole of α2M; and 3) these reactions result in exposure of ∼4 SH groups per α2M molecule (probably one per subunit) by cleavage of non-peptide bonds which are readily susceptible to nucleophilic attack.
A New Method To Determine The Binding Of Factor X To Phospholipid Bilayers
Authors: Tans G , Rosing J , Dieijen G V , Rijn J V , Hemker H C
Published: July 2018

The binding parameters (dissociation constant and the number of binding sites) for factor X binding to phospholipid bilayers in the presence of Ca-ions can be determined using the factor X-activating protein present in Russel’s Viper Venom (RVV-X). It is shown that RVV-X is only able to activate factor X molecules that are not bound to the phospholipid bilayer. Therefore, from the observed rates of factor X activation by RVV-X measured in the presence and absence of phospholipid the amount of factor X not bound to the phospholipid bilayer can be calculated. When the concentration of factor X added is known the bound factor X can be calculated and the binding parameters for factor X are obtained from a Scatchard plot. The technique presented here offers an advantage over other techniques published thus far. It is rapid and simple as compared to the Hummel andDryer technique. There is also an advantage over techniques using light scattering. Since the technique presented here is not hampered by aggregation of vesicles it is possible to study the binding of factor X to vesicles containing a high mole fraction of PS and at high CaCl2 concentrations.

The binding parameters for factor X binding to vesicles of a mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) are dependent on the amount of negatively charged phospholipid present. At increasing mole fractions of PS factor X binds more tightly to the vesicles and also the number of factor X binding sites increases. However, at high amounts of PS (60%) the number of sites present decreases dramatically which is likely due to aggregation of the vesicles. Binding to vesicles composed of a mixture of phosphatidylgly- cerol (PG) and PC is considerably weaker whereas the lumber of sites present is about the same. Prothrombin and factor X compete for the same binding site as was determined by competition experiments. Our data will be discussed with respect to the mode of interaction of vitamin-K dependent clotting factors with negatively charged phospholipid interfaces.
The Contribution Of Factor VIII In The Intrinsic Activation Of Human Factor X
Authors: Mertens K , van Wijngaarden A , van Tilburg N H , Bertina R M
Published: July 2018

In the intrinsic pathway, factor X is activated by a complex consisting of activated factor IX, factor VIII, phospholipid and calcium ions. The contribution of each of this components has been estimated by recombination of purified human coagulation factors, and measurement of the initial rate of factor Xa formation using a spectrophoto- metric assay.

In the absence of phospholipid and factor VIII, factor X is activated at a low, but significant rate. Addition of either phospholipid or activated factor VIII stimulates the initial rate of this reaction 250 and 500 fold, respectively; addition of both cofactors stimulates more than 100,000 fold. The activation of factor VIII by factor Xa or by thrombin is essential for the stimulating effect of factor VIII. Factor VIII complex, containing both VIIIR:WF and VIII:C, and purified factor VIII:C behave identically in this system; purified factor VIIIR:WF does not influence the rate of factor Xa formation.

The same kinetic approach was used for the characterization of preparations of abnormal factor VIII, obtained from plasma of haemophilia A patients.
New Evidence For An Adp-Independent Mechanism Of Thrombin Induced Platelet Activation
Authors: Figures W R , Colman R F , Niewiarowski S , Morinelli T , Wachtfogel Y , Colman R W
Published: July 2018

The adenosine nucleotide analog, 5’fluorosulfonylbenzoyl adenosine (5’FSBA) is a potent inhibitor of ADP induced platelet shape change, aggregation, and fibrinogen binding and covalently incorporates into a single membrane polypeptide (Mr=100,000). To evaluate the role of released intracellular ADP in thrombin induced aggregation and fibrinogen binding, the effects of 5’FSBA on these events were studied. Following incubation with 5’FSBA, the platelets became unresponsive to ADP but were aggregated by purified human thrombin (0.2 U/ml). However, the aggregation was slower and less extensive than that of untreated control platelets. Binding of 125I-fibrinogen to control platelets occured after exposure to ADP or after exposure to thrombin in the presence of hirudin to inhibit clotting. Binding of 125I-fibrinogen was absent in platelets treated with 5’FSBA after exposure to ADP and diminished after incubation with thrombin (0.2 U/ml, 5 min.).

The effect of thrombin on the single platelet membrane polypeptide modified by 5’FSBA was examined by SDS-PAGE. Thrombin treatment (0.5 U/ml, 30 min.) of cells labeled with (H)-5’FSBA resulted in partial cleavage of this polypeptide as evident by a decrease in the (H) FSBA-modified polypeptide in the thrombin treated samples as compared to controls. Unlike chymotrypsin activation of platelets, no fragment of the protein at 75,000 daltons was associated with the membranes of thrombin treated cells. The results of these experiments indicate that thrombin induced platelet aggregation and fibrinogen binding depend on both ADP binding which is blocked by 5’FSBA and the proteolytic cleavage of the same 100,000 dalton membrane polypeptide cleaved by chymotrypsin.
Association Of α-Actinin With The Plasma Membrane Of Human Blood Platelets
Authors: Sixma Jan J , Schiphorst Marion E , Jockusch Brigitte H
Published: July 2018

The association of α-actinin with the plasma membrane of human blood platelets was investigated using immuno- adsorbed antibodies against α-actinin from porcine skeletal muscle and against actin from chicken gizzard. Platelet membranes enriched 10 times in radiolabelled wheat-germ- agglutinin were prepared utilizing the polylysine-bead technique of Cohen et al (1977). Immunofluorescent staining showed the presence of actin and α-actinin associated with these membranes.

Platelets were ectolabelled with lactoperoxydase-iodina- tion. A crude membrane preparation was solubilized with Triton X 100, and incubated with anti α-actinin or appropriate control antibodies. The formed immune complexes were bound to Protein A-Sepharose beads, and subjected to 2-D- electrophoresis with i.e.f. in the first dimension, α-actinin from these membranes was not radiolabelled, had an apparent mol wt of 10U kdaltons and a pi of 6.7, similar to an α-actinin preparation from porcine skeletal muscle that was investigated in parallel. The location of α-actinin in the 2-D pattern was different from that of glycoprotein III.

No specific glycoprotein was found bound to α-actinin under the experimental conditions. Treatment of membranes with non-ionic detergents and ‘protein perturbants’ indicated that α-actinin is an ‘extrinsic protein’. These data indicate that α-actinin as well as actin are closely associated with the plasma membrane, linked by electrostatic interaction. Both actin and α-actinin were not exposed on the outside in a resting platelet.
Von Willebrand Factor And Platelet Deposition On Artificials Surfaces
Authors: Maisonneuve P , Pusineri C , Farges J P , Sultan Y
Published: July 2018

Deposition of purified 125I. Von Willebrand factor (VWF) and 51Cr platelets on well characterized artificial surfaces (Rhone Poulenc Lab.) were studied in the in vitro circulcti.cn system of Baumgartner. Free silica silicone rubber, cationic and anionic polyelectrolyte complexe (PEC) were directly synthetised on the central rode of the perfusion chamber. Concentration of 125I VWF varying from 80 ug/ml to 1,5.103 ug/ml were circulated for 10 mn at a shear rate of 800 sec-1 On all surfaces VWF deposition is correlated with the VWF concentration in the circulating solution. With regard to the nature of the surfaces : whatever the concentration of VWF,silicones fixed hundred times less than PEC and among PEC the positively charged surfaces fixed 3 times more VWF than negatively charged PEC.

Similarly 51Cr platelets resuspended in whole blood were studied in the same system at the same shear rate after 10 mn of circulation. It was found that platelet adherence on the various types of surfaces paralleled VWF deposition. In contrast no correlation between 125I Fibrinogen and platelet fixation on the surfaces was found.

In another series of experiments the role of VWF on platelet adhesion to surfaces was tested. It was observed that fixa- tion of normal platelets resuspended in the blood of a severe VW patient was identical to the fixation of platelets resuspended in normal blood. And this was verified after 1,
3, 5 and 10 mn of circulation, assuming that platelet fixation, at least in these experimental conditions, is independent of the presence of VWF in the system and that the plateau is reached before the first minute of contact. It is more likely that the amount of VWF, fibrinogen and the number of platelets deposited on a surface are related to the intrinsic properties of this surface and characterize the surfaces. These results showed that, platelets and VW factor do not interact on artificial surfaces as it has been demonstrated for the subendothelium of the vessel wall.
Interaction Of Lectins With Human Platelet Membrane Glycoproteins In Crossed Immunoelectrophoresis
Authors: Zonneveld G T E , Roozeboom R , Sturk A , ten Cate J W
Published: July 2018

Plasma membranes of human blood platelets contain a large number of glycoproteins (GP). It has been shown that these GPs play a major role in platelet function. Our aim is to isolate and characterize the GPs. Isolation can be achieved oy affinity chromatography. Crossed immunoelectro-phoresis CIE) was found to be an easy technique to test the usefullness of certain lectins. Rabbits were immunized with bound material of a Lens culinaris column. The antiserum obtained gave 2 precipitates in CIE against solubilised membranes. The slow moving precipitate consisted of GP IIb and IIIa. Several lectins were tested in the intermediate gel technique. Wheat Germ Agglutinin, soybean lectin and Riccinus communis 60 (group 1) interacted with the slow moving precipitate, while Pisum sativum, Ulex Europeus and leucoagglu- (group 2) only showed interaction with the fast precipitate. Riccinus communis 120 affected both precipitates. Helix pomatia and limulus lectin were without effect. A combination of affinity chromatography with a lectin of group 1 and a lectin of group 2 seems useful for isolation of GP IIb and IIIa.

The interaction of lectins of different sugar specificity with the GPs in CIE are indicative for their lectin binding characteristics.
The Effect Of A Vasoactive Substance On The Fibrinolytic Activity Of Varicotic Veins And Experimental Thrombosis
Authors: Haas S , Von Eckstädt A Vitzthum , Wriedt-Lübbe A , Söhnchen R , Lill G , Blümel G
Published: July 2018

Benzaron (2-Äthyl-3(4’-hydroxy-benzoyl)-Benzofuran)is used as a vasoactive substance in phlebology. In order to study its effect on the fibrinolytic activity of varicotic veins and thrombosis,two studies were performed.

Study 1: 60 patients with stripping operations were randomly divided into two groups. One group was treated with benzaron 200 mg t.d.s.for two weeks,whereas the control group did not get any medication. Intraoperatively,a one cm segment was taken from the varicose vein and a one cm segment was excised from the vena saphena magna. The tissue specimens were shock frozen and later on,the fibrinolytic activity was determined by fibrinolysis autrography according to TODD.The intensity of lysis was estimated according to the classification of PANDOLFI.

After treatment with benzaron,the fibrinolytic activity of the intima of the varicotic veins and the vena saphena magna was significantly increased in comparison with the control group.

Study 2: An experimental thrombus formation was induced by endothelial lesion..This was performed by exposure of a one cm segment of the left jugular vein to hypothermia of-10°C for two minutes. The rats were pretreated with benzaron or placebo.24 hours later the thrombus size was determined by measurement of the weight using an analytical balance.

In comparison with the control group the thrombus size was markedly reduced in the benzaron group.

Conclusion:The reduction of venous thrombus formation after treatment with benzaron might be due to the enhancing effect of the substance on the fibrinolytic activity of the vessel wall. These findings should be considered in therapy of patients with reduced plasminogen activators in veins,e.g.in patients with varicosis.
Complex Formation Between Prothrombin And Staphylocoagulase
Authors: Lindhout M , Hendrix H , Hemker H
Published: July 2018

Staphylocoagulase is a protein secreted by certain strains of Staphylococcus aureus. Addition of the extracellular protein to a human prothrombin preparation results in the generation of a thrombin-like activity.

Our investigations on the prothrombin-staphylocoagulase interaction was undertaken in order to establish the kinetics of the complex formation, the mechanism of activation of prothrombin by staphylocoagulase and the enzymatic properties of the complex.

Evidence for the formation of a equimolar complex between prothrombin and staphylocoagulase was obtained fromgelelec- trophoresis and titration experiments. The procedure,in order to study the kinetics of the complex formation, made use of a coupled assay in which D-Phe-Pip-L-Arg-p-nitroanilide hydrolysis by the complex was monitored. The second rate constant of the complex formation, as determined from pseudofirst order kinetics, is 4×l06sec-1M-1. The dissociationconstant of the complex was determined to be orders of magnitude less than 10-11M. Using 3H-DFP, no exchange was observed between prothrombin in the complex with free prothrombin. Analysis of the complex by gelelectrophoresisin the presence of Na-dodecyl sulfate and mercaptoethanol shows that proteolysis cannot account for the generation of thrombin-like activity. Small amounts of degradationproducts (prethrombin 1) were found.

Thrombin-like activity was generated as a result of the interactions between staphylocoagulase and human prethrombin 1, human prethrombin 2 and bovine prethrombin 2. Indications for an interaction between staphylocoagulase and thrombin were obtained from experiments with anti-thrombin III and hirudin. Identical enzymatic properties of thrombin and the prothrombin-staphylocoagulase complex were observed towards small synthetic substrates and fibrinogen. No activity of the complex towards factor V, factor VIII and platelets was found.
Studies On The Relationship Between Factor VIII Related Antigen (VIIIRAg) And Factor VIII Clotting Antigen (VIIICAg) By Immunoelectrophoresis And Autoradiography Using I125 Anti VIIICAg
Authors: Davies B L , Furlong R A , Peake I R
Published: July 2018

In order to determine the relationship between factor VIII related antigen (VIIIRAg) and factor VIII clotting antigen (VIIICAg), a technique has been developed which involves preincubation of plasma and sera samples with I125 anti VIIICAg antibodies, prior to Immunoelectrophoresis against anti VIIIRAg. Autoradiography of the VIIIRAg immunoprecipitin line indicates the presence or absence of anti-VIIICAg associated with the factor VIII complex. In normal citrated plasma, the presence of 125I radioactivity associated with the precipitin arc suggests that VIIIRAg and VIIICAg are associated. The lack of radioactivity associated with VIIIRAg in serum suggests complete dissociation of VIIIRAg and VIIICAg. This pattern was also seen in recalcified normal plasma and in purified factor VIII concentrates which had previously been incubated with purified thrombin, the radioactivity in the latter case being concentration dependent, with complete dissociation of VIIIRAg and VIIICAg at levels >0.1 u/ml thrombin. This result suggests that the dissociation of VIIIRAg and VIIICAg in serum may be due to the action of thrombin in the clotting process. In VIIICAg positive (CRM+ve) plasma samples, radioactivity was associated with the top of the VIIIRAg rocket only, which is similar to that seen for heparinised normal plasma. Reduction of radioactivity occurred with increasing ionic strength when high molar NaCI was incubated with normal citrated plasma. This technique is useful in studying the relationship between VIIIRAg and VIIICAg, and in the identification of CRM+ve haemophilia.
Reduced Arteriosclerotic Plaque Thrombus Formation
Authors: Dujovny M , Kossovsky N , Loubeau J-M , Segal R , Nelson D
Published: July 2018

Sixty surgical patients with cerebrovascular accidents and angiographically diagnosed carotid plaques were divided into two groups: .1) pre-treatment with aspirin (650mg/day) and dipyridamol (25mg/tid) (42 patients); 2) no treatments (18 patients). The control group consisted of 10 autopsy of patients dying for unrelated cause.

SEM examination of the endarterectomy plaque revealed intact red blood cells in 12 of 42 pre-treated and 18 of 18 untreated specimens; degenerating red blood cells and cell fragments (24/42, 18/18); fibrin (24/42, 18/18) and white blood cells (15/42, 18/18). These were frequently enmeshed in large surface thrombi. Cholesterol crystals (6 μm) were matted on the plaque surface (12/42, 15/18), and spherical globules averaging 3 to 10 μm sporadically blanketed the surface (6/42, 6/18). Plaque from the asymptomatic controls was smaller and less thrombotic. Energy dispersive analysis x-rays (EDAX) of the inner surface of all 70 specimens (50 years old +) revealed surface calcium deposits and nodules in 18 of the surgical cases (12/42, 6/18), and diffuse calcium deposits in 6 controls.

Treatment with aspirin and dipyridamol significantly reduces plaque thrombosis.
Peripheral Vascular Disease: Comparative Ultrastructure Of Veins, Valves And Arteries
Authors: Reddick R L , Griggs T , Romanenko A , Brinkhous K M
Published: July 2018

Ultrastructural changes in the coronary and cerebral arteries in thromboatherosclerosis have been well documented. Similar detailed studies in peripheral vascular disease (PVD) have not been described. Since PVD is frequently complicated by venous thrombosis, a comparative study of the arterial and venous pathology in patients with occlusive peripheral atherosclerosis was done. Anterior tibial artery and accompanying veins from seven cases were studied by light, transmission and scanning microscopy. Endothelial cell morphology was abnormal in all arteries. Cells contained lipid inclusions, prominent lysosomes, and cytoplasmic filaments were disorganized. Cell junctions were altered. Endothelial cells from both the vein wall and valve were morphologically normal. Basement membrane was reduplicated in both arteries and veins. In some arteries, the basement membrane was partially obscured by the atheromatous changes. Elastic lamina in veins, valves and arteries was fragmented. Smooth muscle cells of the arterial intima has significant morphologic alterations. The cells had a disorganized pattern and contained glycogen granules, lipid droplets and lysosomes in association with lipid. The filamentous pattern was disorganized and many cells appeared devoid of a basement membrane. Pinocytic vesicles were clearly evident. Smooth muscle cells comprising vein wall and valve were unremarkable. Collagen in arteries, veins and valves was haphazardly arranged with variation in fibrillar size. In some arteries, fibrous type collagen was found. Irregular, smudged collagen was present in all vessels examined. Calcification was noted in arteries only. These findings show a) a diversity of transmural alterations in arterial wall and b) ultrastructural changes in collagen in venous wall. The former is associated with thrombotic events, the latter not.
The Effect Of Dexamethasone On Basement Membrane Produced By Cultured Human Endo Thelial Cells
Authors: Piovella F , Ricetti M M , Almasio P , Ascari E
Published: July 2018

Human endothelial cells from umbilical cord vein were grown to confluence on glass coverslips placed in Petri di= shes, in the presence or absence of dexamethasone. After ten days in culture, the cell monolayers were washed and treated with 0,5% Triton X lOO in Hank’s solution , The detergent-treated slides were then washed in Hank’s and examined by indirect immunofluorescence for fibronectin utilizing monospecific antibodies. Basement membranes produced by dexamethasone-treated cells showed a more intense fibrillar network of extensively overlapping fluorescent fibers if compared with controls.

The effect of these pre-formed basement membranes on cellular spreading was subsequently studied. Human endothelial cells , previously grown in culture medium with out steroids, were detached by trypsin-EDTA solution,washed and cultured on the slides covered with the extracel lular matrix pre-formed in the presence or absence of dexamethasone. The higher fibronectin content of basement membranes pre-formed in the presence of dexamethasone allowed a more rapid attachement and a better spreading of endothelial cells if compared with control pre-formed basement membranes.
Intraplatelet And Plasma Serotonin Level In Normal Subjects And In Patients With Cerebrovascular Diseases
Authors: Kita E , Horiike N , Kyoi K , Utsumi S , Umegaki K
Published: July 2018

Intraplatelet and plasma serotonin(5-hydroxytryptamine:5- HT) was assayed spectrophotometrically using O’phthaldialdehyde as the fluorescence inducer depend on the method of Durmmond and Golden, in 69 healthy volunteers and in 69 patients with various kinds of cerebrovascular diseases such as acute cerebral thrombosis within 72 hours from onset (13 cases), chronic cerebral thrombosis during 2 to 48 months from onset(27), and T I A(ll), RIND(2), S A H(5), intracerebral hematoma(7) and recurrent cerebral thrombosis under treatment.

In normal controls, intraplatelet 5-HT level had the tendency of gradual decrease with the aging, on the other hand, plasma level of 5-HT in same subjects showed slight increse in elder groupe.

In acute complete thrombosis, intraplatelet 5-HT level was significantly low compared with age matched controls with slightly elevated level in plasma. In the reversible stroke such as T I A and RIND, intraplatelet level was low as same as complete cases, but plasma level was within normal range. In other C V D, none of them showed significant change of 5-HT level either in platelet or plasma.

Recurrent cerebral stroke patients under various periods of different treatment had enormous high level of intraplatelet 5-HT level with normal value of plasma.

Above mentioned results suggest that in acute thrombusforming state, intraplatelet 5-HT level is reduced or we may say there circulate “exhausted” platelets in the plasma.
5'-N-Ethylcarboxamidoadenosine (NECA) Is A Potent, Stereospecific Inhibitor Of Human Platelet Aggregation
Authors: Cusack N J , Hourani S M O
Published: July 2018

Adenosine is a vasodilator, and also inhibits platelet aggregation apparently by acting at an external membrane receptor to increase levels of platelet cyclic AMP. Certain analogues of adenosine retain activity as vasodilators, and also inhibit platelet aggregation by raising levels of platelet cyclic AMP. NECA is an extraordinarily potent vasodilator, so its effects on human platelet function were tested.

NECA (1 μM) inhibited human platelet aggregation induced by ADP, 5-HT, thrombin and adrenaline mere powerfully than adenosine (1 μM). NECA, even at micrcmolar concentrations, was 5 to 10 times more potent than adenosine as an inhibitor of aggregation induced by ADP (5μM or 200μM) or adrenaline (200μM). NECA (Ka = 0.95μM) caused increases in levels of platelet cyclic AMP, which could be competitively inhibited by theophylline (Ki = 8μM), an adenosine receptor antagonist. However, here NECA was only about
1.3 times more potent than adenosine (Ka = 1.2μM). The effects of NECA, like those of adenosine, were completely stereospecific, the L enantiomer of NECA being inactive. NECA (10μM) did not prevent inhibition of PGE1-stimulated adenylate cyclase by ADP (5 μM).

NECA is the most potent analogue of adenosine yet tested on human platelets, and is the first example of a 5' modification to retain significant inhibitory potency.
Suppression Of Platelet Aggregation By A New Beta-Adrenergic Blocker
Authors: Small M , Douglas J T , Orr Morna , Lowe G D O , Forces C D , prentice C R M
Published: July 2018

Beta-blockers have been reported to have a variable effect on a number of haemostatic parameters. In view of their possible protective action in coronary artery disease, we studied the effect of the new long-acting beta-blocker, carteolol, on platelet function, coagulation and blood viscosity in 10 healthy male volunteers. Following carteolol (5 mg. orally) blood was taken at 2, 5 and 24 hours and we were able to demonstrate a significant inhibition of platelet aggregation to ADP, adrenaline and collagen (p< 0.05) at 5 hours post ingestion, but not at 2 or 24 hours. Platelet release as measured by plasma levels of beta-thromboglobulin, platelet count, fibrinopeptide A, whole blood viscosity and plasma viscosity was also unaltered.
Roles Of Cyclic Nucleotides In The Inhibition Of Platelet Function By Nitroprusside And By Ascorbate
Authors: Davidson M M L , Haslam R J
Published: July 2018

The effects of nitroprusside and of ascorbate on the collagen-induced aggregation of washed human platelets and on the associated release of dense granule constituents were correlated with their effects on platelet cyclic GMP and cyclic AMP, which were measured either by radioimmunoassays or prelabelling methods. Nitroprusside at concentrations from 1 to 400 μM increased platelet cyclic GMP from 6 to 100-fold (maximum value approx. 50 pmol/109 platelets) and at concentrations above 10 μM also increased cyclic AMP about 2-fold (maximum value approx. 36 pmol/109 platelets). Platelet cyclic GMP reached a peak after an incubation period inversely related to the nitroprusside concentration and then declined. Collagen, which increased platelet cyclic GMP about 2-fold, enhanced the effect of nitroprusside on cyclic GMP but not cyclic AMP. Freshly prepared ascorbate (10 mM) increased platelet cyclic GMP about 8-fold. Storage of the ascorbate at pH 7 or simultaneous addition of 5 μM CuCl2 potentiated its action to give 15 to 20-fold increases in cyclic GMP and small increases in cyclic AMP. The results suggested that oxidation of the ascorbate was involved in these effects. In all the above studies, increases in platelet cyclic GMP greater than 6 to 10-fold were associated with measurable increases in cyclic AMP and with inhibitions of collagen-induced platelet responses that roughly correlated with the cyclic nucleotide changes. Addition of 100 μM 2',5'-dideoxyadenosine (an inhibitor of adenylate cyclase) blocked increases in platelet cyclic AMP but did not affect increases in cyclic GMP; this compound also decreased (but did not abolish) the inhibitory effects of nitroprusside and of ascorbate + CuCl2 These findings suggested roles for both cyclic GMP and cyclic AMP in mediating the inhibitions of platelet function by nitroprusside and ascorbate.