bluuebunny/crossref_metadata_2025 huggingface dataset:
Ddavp-Induced Alterations In The Multimeric Composition Of Factor Viii/Von Willebrand Factor In Normal And Von Willebrand’S Disease Plasma
Authors: Ruggeri Z M , Lombardi R , Mannucci P M , Zimmerman T S
Published: July 2018

DDAVP is known to induce increases in plasma FVIII/von Willebrand Factor (FVIII/vWF) concentration by release of
tissue stores. We have now found that the multimeric composition of plasma FVIII/vWF alters following DDAVP administration to resemble that seen in platelets. Multimers were resolved by thin-layer SDS-agarose and SDS-agarose acrylamide electrophoresis and were identified by reaction of the fixed gels with 125I-laben ej antibody followed by autoradiography. Fifteen minutes after DDAVP infusion, larger multimers appeared in normal plasma than were present during the resting state. These larger multimers were also present in resting normal platelets, suggesting that DDAVP causes their release from tissue stores. In Type I von Willebrand s disease (vWd), there is a decreased quantity of circulating FVIII/vWF but the multi meric composition is normal in plasma and platelets. The response to DDAVP was qualitatively similar to that in normal individuals. In Type IIA vWd large multimers are absent both from plasma and platelets. Even though DDAVP induced a threefold rise in FVIII/vWF plasma concentration, these large forms did not appear. In 11B vWd only intermediate and small multimers are present in plasma but there is a normal complement of multimers in platelets. Following DDAVP there was a rapid correction of the plasma multimeric composition so that it was identical with the pattern seen in normals after DDAVP. However, this correction was transitory and the abnormal basal pattern soon reappeared. These disparate changes in plasma FVIII/vWF multimeric composition following DDAVP suggest that platelet FVIII/vWF is representative of releasable tissue stores. They also provide insight into the variable hemostatic response to this agent and suggest different pathogenic mechanisms for the vWa subtypes.
Plasma And Urine Betathromboglobulin Concentration In Deep Vein Thrombosis
Authors: Turpie A G G , de Boer A C , Han P , Butt R , Genton E
Published: July 2018

To investigate the involvement of platelets in the genesis of deep vein thrombosis (DVT) plasma and urine betathromboglobulin (BTG) concentration (normal; plasma 2 8 ± 8 ng/ml; urine 0.18 ± 0.18 ng/ml; mean ± SD) was measured serially in high-risk postoperative patients undergoing surveillance with 125Iodine-fibrinogen leg scanning. BTG cone, was consistently increased postoperatively but returned to normal within 2-3 days. In 30 patients with negative 125I-fibrinogen leg scans, BTG remained normal. In the 10 patients who developed DVT, BTG cone, became elevated before the 125I-fibrinogen scan became positive and remained elevated for several days and then returned to normal. The pattern of BTG elevation was not related to anticoagulant therapy. To evaluate BTG in the diagnosis of DVT, plasma (n=l52) and urine BTG (n=72) were measured in symptomatic patients referred for venography. In the patients with positive venograms, mean plasma BTG cone, at presentation (75 ± 77 ng/ml) and mean urine BTG cone.(0.63 ± 0.77 ng/ml) were significantly elevated compared with plasma BTG (42 ± 36 ng/ml) or urine BTG (0.15 ± 0.13 ng/ml) in patients with negative venograms (p< 0.005) and compared with normal values (p < 0.005). Mean plasma and urine BTG cone, were not different between patients with calf vein thrombi compared with proximal vein thrombi. There was a significant correlation between plasma and urine BTG cone. (r=0.68, p<0.005). The sensitivity, specificity and accuracy of elevated plasma BTG (>50 ng/ml) at presentation for the diagnosis of DVT were 35%, 80% and 64% respectively and for urine BTG(>0.54 ng/ml) were 37%, 100% and 74%. The results indicate that platelet release occurred with DVT but in most instances was limited to the early phase. BTG cone, may have value for surveillance but measurement of BTG is of little value for the diagnosis of DVT in symptomatic patients.
Radioimmunoassay Of Fragment E In Suspected Pulmonary Embolism
Authors: Duffy M J , Farrell R J , Duffy G J
Published: July 2018

Previous investigators have reported that radioimmunoassay of fragment E (FgE) can distinguish between symptomatic deep venous thrombosis and other conditions giving a similar clinical picture. Elevated serum FgE levels have been reported in pulmonary embolism, but these have not been compared with non-thromboembolic lung disorders. In this study serum FgE was measured in 44 patients undergoing lung scans for suspected pulmonary embolism and in 12 patients with pneumonia.

Serum FgE levels greater than 250 ng/ml were found in 2/22 patients with normal lung scans, in 9/12 with pulmonary embolism (PE), in 3/6 with chronic obstructive airways disease (COAD), in none of 4 patients with congestive cardiac failure (CCF) and in 5/12 with pneumonia. Mean serum FgE levels in the above groups.

In this series, serum FgE did not discriminate between pulmonary embolism and non-thromboembolic lung diseases. Radioimmunoassay of FgE may have some value in screening for thromboembolism but it is not sufficiently specific tobe used as an indication for anticoagulant or thrombolytic therapy.
Platelet Calmodulin Mediates The Calcium Dependent Activation Of Factor XIII
Authors: Kahn D , Crawford N , Cohen I
Published: July 2018

Transglutaminases are ubiquitous in cells and require calcium for their activation. The factor XIII zymogen, present in plasma and in the platelet cytosol, requires for its activation both a limited proteolytic activity on the catalytic subunit,“a”, and, in the use of the plasma enzyme, calcium for dissociating subunit“a” from the carrier subunit“b”. Calcium is also necessary for exposing the reactive sulfhydryl group. We have recently suggested a role for the platelet factor XIII in the generation of calcium-dependent cross-linking processes in platelets. Since calmodulin is present in considerable amounts in the platelet cytosol and is known to regulate the activity of various calcium-dependent enzymes and cellular reactions, we have investigated its possible role in factor XIII activation. Since the“a” subunit of platelet factor XIII is identical to its plasma counterpart, the more easily purified plasma zymogen was used. The effect of calmodulin on the two calcium-dependent steps of factor XIII activation was investigated following thrombin-stimulated hydrolysis of the“a” subunit. Platelet calmodulin was found to enhance by at least 3 fold the calcium-dependent unmasking of the reactive sulfhydryl groups which were titrated with 14C-iodoacetamide. Calmodulin also enhanced by at least 4 fold the calcium-dependent dissociation of the b subunit from its complex with the thrombin-hydrolyzed“a” subunit. The calmodulin mediation of the calcium-dependent steps of factor XIII activation may be important for regulating the factor XIII-dependent cross-linking reactions in platelets and is reminiscent of the calcium-related regulatory role of fibrinogen on factor XIII activation which could prevail in plasma. An investigation of the possible role of calmodulin on other tissue transglutaminases is warranted.
Inactivation Of Purified Human Platelet Cyclic Amp Phosphodiesterase By The Affinity Label 2’-O-Iodohydrin-P-Cyclic Amp
Authors: Grant P G , Colman R F , Sinha A K , Colman R W
Published: July 2018

Cyclic AMP phosphodiesterase (PDE) is a regulatory enzyme in human platelets. Inhibitors of this enzyme raise intracellular cAMP which prevents platelet activation. Little is known about the biochemistry of this enzyme. PDE was isolated from human platelet concentrates by nitrogen bomb cavitation. The specific activity of PDE in cell lysate was 0.064 nmoles cAMP hydrolysed/min/mg protein at 22° , 1 µM cAMP. Eighty percent of the activity appeared in the 100,000 × g supernatant fraction. Chromatography was performed on DEAE cellulose equibrated with 50 mM Tris- acetate pH 6.0, 3.75 mM 2-mercaptoethanol. A linear gradient with a limiting salt concentration of 1.0 M Na acetate separated two peaks of PDE activity. The first had a for cAMP of >100 µM; the second had a Km for cAMP of 5 µM. The lower enzyme was further purified by adsorption on blue dextran Sepharose in 50 mM Tris pH 7.5, 2 mM MgCl2 followed by affinity elution with 1 mM cAMP in the same buffer. These steps resulted in a 1700 purification of the enzyme (113 nmoles/min/mg). The compound 2’-0-iodohydrin- p-cAMP (IH-cAMP) is a cAMP derivative with an alkylating side chain. Incubation of PDE with 5 mM IH-cAMP at 37° resulted in 88% inactivation of the enzyme at 15 hours, compared to a control, with a corrected pseudo-first-order rate constant of 0.144 h-1. When cAMP (100 µM) was included in the inactivation mixture the corrected pseudo-first-order rate constant decreased to 0.064 h-1l. Thus, a 20-fold excess of cAMP protected 56% against inactivation by IH- cAMP. The inhibition was not reversed by gel filtration of the inactivated enzyme which removed IH-cAMP. These results suggest that IH-cAMP reacts with the active site of PDE to irreversibly inactivate the enzyme. IH-cAMP should prove to be a useful tool in understanding the chemistry of the active site of this important enzyme.
Studies On The Mechanism Of Platelet Adhesion To Collagen- Sepharose
Authors: Capobianco J O , Miller M F , Holleman W H
Published: July 2018

Platelet interaction with the collagenous material of damaged vessel walls is one of the primary events leading to cardiovascular disease. In vitro methods and known antiplatelet agents were employed in order to better understand the adhesion and release processes. These processes were quantitated using 51Cr and l4c-serotonin labeled rabbit gel filtered platelets (GFP) and immobilized collagen. Metabolic inhibitors, antimycin-A and 2-deoxyglucose, partially reduced adhesion (45%) while addition of 0.1% glucose reversed their effect. Scanning electron microscope studies indicated that energy depleted GFP bound less tightly to collagen fibers and remained disc shaped. These and other studies concluded that the adhesion process consisted of an energy dependent phase. Membrane active compounds such as the phenylthiazines strongly inhibit adhesion (>70%) at concentrations up to 0.1 mM. Their activity was related to their ability to penetrate the membrane’s lipid layers. Verapamil, a Ca++ channel blocker, was inactive. ADP receptor antagonists affected both adhesion and release to varying degrees. Phosphodiesterase inhibitors and adenyl cyclase activators were most effective in combination in reducing release (>70%) but weak in reducing adhesion (<22%). Studies with 13-azaprostanoic acid (13-AZA), a thromboxane-A2 antagonist (TXA2), indicated that TxA2 generation had very little effect on adhesion or release from adhering platelets. Sulfhydryl modifying agents were effective in reducing both adhesion (>59%) and release (>57%). This report concludes that the platelet-collagen interaction was a multi-component process modified by small organic molecules. Furthermore, it was demonstrated that the release process of adhering platelets was independent of the adhesion process initiated by collagen.
Isolation Of Two Types Of Platelets By Collagen-Sepharose Conjugate And Elucidation Of Their Roles In Adhesion And Aggregation
Authors: Saito Y , Imada T , Matsushima A , Inada Y
Published: July 2018

Approximately 50% of washed platelets from bovine blood adhered to collagen-Sepharose conjugate. They, however, did not adhere to Sepharose itself and Sepharose conjugated with serum albumin and ovalbumin. The extent of adhesion to collagen-Sepharose conjugate was increased neither by decreasing the number of platelets applied, nor by increasing the column volume. Platelets in the normal blood may thus be subdivided into two groups depending on their adhesiveness to collagen-Sepharose, adhesive and non-adhesive cells. The adhesive cells, which were obtained by briefly exposing cells to ADP, did not aggregate themselves, while the non-adhesive cells aggregated quite normally. Furthermore, we were able to reveal that adhesive cells could aggregate only with nonadhesive cells. We propose that these two types of platelets play their respective roles sequentially to ensure the successful formation of thrombus in vivo. The investigation with human platelets is under way.
CA2+ Affects Platelet-Subendothelium Interaction Independent Of F VIII-Vwf
Authors: Sakariassen K S , Sixma J J , Bolhuis P A
Published: July 2018

The effects of Ca2+ in the presence or absence of F VIII-VWF on blood platelet adherence to subendothelium of human arteries were investigated in an annular perfusion chamber according to Baumgartner, using a non-pulsatile perfusion system. The perfusates consisted of citrated blood or reconstituted fluids with washed red blood cells, 51Cr-labelled platelets, and the appropriate plasma replacements. F VIIIVWF was purified from cryoprecipitate by Sepharose CL-4B chromatography and radio-labelled with 125I by the lactoperoxidase tecjiijtique. Platelet adherence was measured by registration of 51Cr count or by morphometry. Free Ca2+ was assayed with a calcium electrode, with a minimum detection limit of 10 µM. A close correlation was observed between the Ca2+ concentration and the haematocrit in normal blood anticoagulated with l/lO vol 110mM Na3-citrate. The plasma concentration of citrate and Ca2+ had mean values of l8 mM and 65 yM respectively. Perfusion for 7 min at a vessel wall shear rate of 1000 s-1 with this blood, produced a monolayer of adherent platelets and only occasionally small platelet aggregates on the subendothelium2+but no thrombi > 5 µM in height. At plasma citrate and Ca2+ concentrations of respectively 40 mM and 25 µM the platelet spreading on the subendothelius^ was impaired. At a citrate concentration of l60 mM and a Ca2+ concentration below the detection limit, the initial contact between platelets and subendothelium was abolished. In perfusion fluids in which plasma was substituted by a human albumin solution (HAS), the adherence at Ca2+ concentrations above 10 µM was higher in the presence of F VIII-VWF than in its absence. However, at a Ca concentration below 10 pM induced by addition of citrate (160 mM in HAS), the adherence yith and without F VIII-VWF was abolished. The binding of 125I F VIII-VWF to the subendothelium was unchanged at levels below 10 µM Ca2+.

These data indicate that the various steps of pikelet interaction with subendothelium require specific Ca2+ concentrations. The effect of F VIII-VWF is independent of this.
Platelet-Vessel Wall Interactions In Experimentally Induced Hypercholesterolemia
Authors: Tremoli E , Aqradi E , Socini A , Petroni A , Galli C
Published: July 2018

A simple method was developed to study platelet-vesseln wall interactions based on the perfusion of platelet rich plasma (PRP) through isolated segments obtained from the aorta of the same animal. The inhibition of aggregation of the perfused PRP, indicating prostacyclin (PGI2)-like material production by aortic wall, was quantified. The effect was not present when normal PRP was perfused through the vessels obtained from aspirin-treated animals. This experimental model was used in the study of platelet-vessel wall interaction in normal (N) and hypercholesterolemic (HC) rabbits (one month on a high cholesterol -2% W/W - diet).

In the HC animals increased aggregating response coupled with reduced platelet sensitivity to the inhibitory effect of exogenous PGI2 was observed. When PRP of the two groups of animals was perfused through their own aortas, the inhibition of aggregation was significantly lower in HC samples, suggesting ever lower aortic production or lower sensitivity to the inhibitory effect of PGI2-like material in treated animals. In addition a lower inhibition of platelet aggregation occurred after perfusion of PRP from HC animals through aortas of N rabbits, indicating a decreased platelet sensitivity to the inhibitory effect of PGI2-like material released.

It appears that in experimental HC rabbits, platelet aggregation and their sensitivity to the antiaggregatory effect of PGI2 are significantly affected. In our experimen tal conditions, production of PGI2-like material in the aorta is not reduced, but the overall outcome of plateletvessel wall interaction is a reduced inhibition of platelet aggregatory response.
Action Of Ticlopidine On The Oxygenated Metabolism Of Arachidonic Acid In The Mouse Peritoneal Macrophages
Authors: Rigaud M , Rabinovitch H , Durand J , Breton J C , Rigaud G
Published: July 2018

When ticlopidine is added to macrophages cultures, in the absence of exogenous arachidonic acid, there is a production of both“prostanoids” and“eicosanoids” in the culture medium. These products have been measured using glass capillary gas chomatography prior to multiple ion mass spectrometry. The quantitative determinations are made 5, 15, 25, 35 and 45 minutes after the drug was added to the macrophages cultures. The three drug concentrations used (10-4M, 5.10-5M and 2.5 10-5M) induce a liberation of 6-keto-PGF1α in the culture medium. As in our system 6-keto-PGF1α seems to be the major metabolite of PGI2, ticlopidine is likely to act by releasing important quantities of PGI2 in macrophages. These results suggest an increase of liberation of the endogenous arachidonic acid from the membrane phospholipids of the macrophages or a lack in the acyltransferase system of the cell membranes. The lipoxygenasic pathway was also studied. When ticlopidine is added to macrophages, two products are liberated: . 12-HETE as measured by single iondetection . and 10-hydroxy-ll-12-epoxy, -5, 8, 14-eicosatrienoic acid which comes from an internal rearrangement of the 12-HPETE. In these results, there is a discrepancy between the fact that ticlopidine increases the concentration of 12-HETE and surely its precursor the 12-HPETE and the fact that the synthesis of PGI2 is not inhibited by these high concentrations of hydroperoxide. To understand this phenomenon we studied the production of hydroperoxide when arachidonic acid is incubated with soybean lipoxygenase. When ticlopidine (10-4M) is added to the reaction mixture (AA + soy lipoxygenase) there is an increase of the initial rate and an extent of the reaction as if the enzyme irreversible deactivation was postponed. Ticlopidine could act by suppressing the classical inhibition of PGI2 synthetase by hydroperoxides, in particular 12-HPETE and 15-HPETE, both produced by mammalian cells.
The Association Of Thrombin With Fibrin And Its Solubilization During Plasmic Digestion
Authors: Francis Charles W , Marder Victor J , Barlow Grant H , Martin S Eric
Published: July 2018

It has previously been demonstrated that thrombin binds to fibrin during clot formation. We have studied the nature of this association and the fate of bound thrombin during fibrin degradation by plasmin. Approximately 10% of human thrombin used to clot human fibrinogen bound to the fibrin and could not be removed by washing in buffers of physiologic pH and ionic strength. Plasmic digestion released thrombin into the lysate where it retained enzymatic activity, as measured by chromogenic and clotting assays. The fibrin was degraded to a group of unique degradation complexes of molecular weight between 230,000 and greater than 800,000. The active thrombin was present in the lysate both as free enzyme and bound to certain of these complexes, primarily those larger than 230,000 daltons. At low initial thrombin concentrations, most of the active enzyme released during plasmic digestion was bound to fibrin derivatives, while at higher initial thrombin concentrations, most was released as free enzyme. This suggests that there are at least two types and/or sites of association between thrombin and fibrin. The presence of thrombin on fibrin clots and bound to soluble fibrin derivatives may have pathophysiologic importance in the propagation of thrombi and in the development of hypercoagulable states.
Biosynthesis Of Human Fibrinogen In A Cell Free System
Authors: Uzan G , Ardaillou N , Larrieu M J , Kahn A , Marguerie G
Published: July 2018

In order to investigate the early events involved in the biosynthesis of human fibrinogen mRNA was isolated from human liver and translated in a rabbit reticulocyte system. The translation was carried out in the presence of 35S-Methionine. Neosynthesized fibrinogen was isolated by immuno- adsorption on microcolumns of anti-fibrinogen IgG coupled to ultrogel and analyzed by vertical slab polyacrylamide gel electrophoresis followed by autoradiography of the gel. Additional identification was achieved by immunological competition with non labeled plasmatic fibrinogen. The results indicated that each polypeptide chain was synthesized separately. The neosynthesized Aα chain exhibited a higher molecular weight that the plasmatic Aα chain. Based on electrophoretic mobility a difference in molecular weight of 2.000 dalton was estimated suggesting the existence of a signal sequence on the nascent Aα chain. The neosynthesized Bβ and γ chains showed lower molecular weight, when compared to the corresponding plasmatic Be and y chains. Since the Bβ and the γ chains are normally glycosylated, the presence of a signal peptide on the nascent chains was not definitely established from electrophoretic mobility alone. In addition total human mRNA were partially fractionated on a sucrose gradient and the different fractions were translated. The precursor of the Aα, Bβ and γ chains were synthesized by mRNA present in the 18 S fraction. These observations established that human fibrinogen was not synthesized as a single chain large precursor and that separate mRNA exist for each polypeptidic chain.
Sequential Plasmin Proteolysis Of The Nh2-Terminus Of The Bβ Chain Of Human Fibrinogen
Authors: Koehn J A , Hurlet-Jensen A , Nossel H L , Canfield R E
Published: July 2018

Recent clinical data have shown that early plasmin proteolysis of human fibrinogen results in the release of fibrinopeptide B (FPB) immunoreactivity into plasma. Upon thrombin treatment this activity increases indicating that the immunoreactivity arises from larger FPB-containing peptides. The presence of degradation products from the Bβ chain may be an index of fibrinogen proteolysis not only in normal hemostasis but also in pathological disorders such as thrombosis and disseminated intravascular coagulation. Therefore the capability of detecting events leading to progressive plasmin degradation at the Bβ chain could be of major diagnostic and therapeutic significance. Toward this goal, we have isolated the various peptides released from human fibrinogen by plasmin proteolysis using HPLC on a reverse phase C18 column. These peptides were identified using amino acid analysis and radioimmunoassays for FPB. Bβ 1-42 was the earliest fragment released during limited plasmin proteolysis. These levels reached a maximum and then began to fall during the course of the digestion. In addition, increasing levels of Bβ 1-21 and of FPB were shown to lag behind the production of Bβ 1-42. Using purified Bβ 1-42, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components, Bβ 1-14 and Bβ 22-42 and a minor component composed of Bβ 15-42. The rate of this reaction is not affected by the addition of hirudin indicating that it is not caused by trace amounts of thrombin. Taken together these data indicate a sequence of events in which Bβ 1-42 is initially cleaved from fibrinogen during plasmin digestion. Free from its parent molecule, Bβ 1-42 can undergo further plasmin attack, preferentially at the 21-22 bond to yield Bβ 1-21 and B8 22-42. Plasmin then attacks Bβ 1-21 at the 14-15 bond to release FPB.
A New, Rapid And Sensitive Determination Of Fibrinopeptide- A (FPA) By A High Affinity Antibody To FPA Antiserum
Authors: Stehle G , Harenberg J , Schmidtgayk H , Zimmermann R
Published: July 2018

The clinical relevance of the determination of FPA is not yet fully recognized because of the still time consuming radioimmunological techniques. Shortening of the incubation times allways lead to a critical loss of the sensitivity of the assay. We present now a modification which provides highly sensitive and reproducable results within 2hrs.

The ethanolic extraction of FPA from plasma was shortened to 20 min including centrifugation. Samples were analysed in triplicates and evaporated at 50°C on microtiter plates. The addition of 0.2μl normal rabbit serum (NRS) lead to a 20% increase of the reaction rate of the FPA antiserum to FPA. A second antibody with high affinity to rabbit immunglobulin i.e. the FPA antiserum improved the maximal binding to 35% after an incubation period of only 10 min. 35-40000 cpm tracer FPA were added to each sample. FPA antiserum, second antibody and NRS were preincubated for 1-24 hrs and then added together with the tracer to the samples. Thus a sensitive standard curve was obtained between 0.16 and 160 ng/ml (12000-600 cpm). The correlation coefficient of this modification to our previously described method was r=0.96 (n=60) The variation coefficient could be improved substantially to 3.1 for low, 4.0 for medium and 4.5% for high FPA. Normal FPA were measured between 0.16 and 2.5 ng/ml (mean 1.4 ng/ml, n=32).

The presented modification of the radioimmunological determination of FPA overcomes the difficulties of previously described methods and provides acurate results within 2 hrs.
Immunochemical Studies Of The Reaction Of Human Desarginyl-Fibrinopeptide B (Desarg-Fpb) With Anti-Fpb Sera
Authors: Butler V P , Tse-Eng D , Nossel H L
Published: July 2018

Fibrin II formation may be essential in thrombosis. Measurement of free FPB is the only direct index of fibrin II formation but is complicated by the fact that carboxypeptidase B rapidly cleaves the COOH-terminal arginine from FPB in human plasma. To facilitate the assay of Desarg-FPB as an index of in vivo FPB release, the immuno-chemical reactivity of Desarg-FPB with anti-FPB sera has been studied. As previously reported, Desarg-FPB was considerably less effective (0.7-17%) than FPB in inhibiting the binding of an 125I-FPB analog by five of six rabbit antisera studied in detail. Surprisingly, Desarg-FPB was 4 to 27 times.more effective than FPB in inhibiting the binding of an 125I-Desarg-FPB analog by the six anti-FPB sera. For example, in the case of antiserum R-28, FPB was 145 times more effective than Desarg-FPB in inhibiting the binding of the 125I-FPB analog but Desarg-FPB was 27 times more effective than FPB in inhibiting the binding of the 125I-Desarg-FPB analog. These findings indicate that the FPB and Desarg-FPB analogs are bound by different antibody populations. We suggest that carboxypeptidase B converts some molecules of FPB-protein conjugates to Desarg-FPB derivatives during the immunization of rabbits. From a practical point of view, the difference in reactivity of the different antisera has enabled the rational selection of anti-FPB sera for use in the assay of Desarg-FPB and its distinction from FPB. With the use of an appropriate antiserum, Desarg-FPB levels have been measured in clinical blood samples and the sensitivity is such that distinction can be made between levels in normal individuals and in disease states.
Fibrinogen Catabolism In The Nephrotic Syndrome
Authors: Fletcher A P , Alkjaersig N , Narayanan M , Robson A M
Published: July 2018

Children with a nephrotic syndrome with renal biopsy findings of either minimal change disease (MCD) or focal glomerulosclerosis (FGS) had been followed serially by plasma fibrinogen chromatographic and other blood coagulation and plasma fibrinolytic assays. Patients with MCD were studied during: 1) relapse without steroid treatment (n=34); 2) relapse on steroid treatment (n=12); 3) early remission on steroid treatment (n=14); 4) late remission on steroids (n=17); and 5) late remission no steroids (n=10). The control group comprised 50 aged matched healthy children. Untreated MCD patients, regardless of disease etiology, showed statistically significant increases in plasma fibrinogen, plasma high molecular weight fibrin(ogen) complexes (average 79 mg/dl compared to 24 ml/dl control) (p < 0.001), in fibrinogen first derivative concentration and α2-macroglobulin, but factor XIII, α1-antitrypsin, plasminogen and antithrombin III concentrations were within the normal range. These findings document pathologically enhanced intravascular fibrin deposition, presumably occurring locally in the glomerulus and with enhanced fibrino- genolysis. Laboratory values reverted toward normal with steroid therapy and were normal in patients in remission whether or not remission was steroid-induced. Patients in relapse showed laboratory findings similar to those of the untreated patient. Laboratory findings in FGS patients in clinical relapse paralleled those found in untreated MCD patients, with disease remission laboratory findings also reverted to normal. It has recently been suggested (J. Ped. Neph. 1:18, 1980) that long-term anticoagulant therapy in FGS patients might prove effective in prevention of glomerulosclerosis and chronic renal failure. Our findings would strengthen the rationale for this therapeutic approach.
In Vivo Studies On The Inhibition Of Coagulation By A Heparin Analogue
Authors: Schmitz-Huebner U , Asbeck F , van de Loo J
Published: July 2018

SSHA, a semi-synthetic heparin analogue belonging to the chondroitin family, was reported to induce considerable anti-Xa activity in vivo being practically inactive in vitro. In a study designed to elucidate further the in vivo effects of this drug, SSHA and sodium heparin from porcine intestinal mucosa were injected subcutaneously into six volunteers on separate occasions over a period of three days in a cross-over trial. Before injection and 2,4,6,8 hours afterwards, the heparin-like activity was measured by means of the APTT, the anti -Xa clotting test and two chromogenic substrate assays. The results show that SSHA mediates both anti-Xa and antithrombin activities in vivo. A comparison between the effects of SSHA and heparin is problematical, due to the heterogeneity of different heparin preparations. Low doses of the analogue (45 mg s.c.) induce proportionally higher and longer lasting anti-Xa activities than higher doses (90 mg s.c.). In an attempt to identify the mediator involved in the anticoagulant activity induced by SSHA in vivo, antithrombin III AT III) was removed from a plasma sample of one the subjects obtaining SSHA injections by immunosorption using Sepharose IVb coupled with antibodies against AT III. The AT III free plasma obtained was found to be devoid of heparin-like activity in the anti-Xa clotting test but it maintained its anticoagulant activity in the APTT assay. When purified AT III was added to this plasma its anti-Xa activity was largely restored. In conclusion, the inhibitory effect of SSHA on coagulation seems to involve at least two mechanisms: a direct one which does not depend on AT III and an indirect one, induced in vivo and mediated by AT III.
Effect Of Heparin On The Thrombin-Antithrombin Reaction Product
Authors: Danishefsky Isidore , Bender Michael S
Published: July 2018

The characteristics of the primary complex (C-l) formed between thrombin and antithrombin in the absence and presence of heparin, were investigated. Each of the complexes were isolated by gel-filtration of the reaction mixture on Sephadex G-100.

Analyses by SDS-polyacrylamide gel electrophoresis showed that thrombin causes the successive degradation of both complexes to lower molecular weight products C-2 and C-3, respectively. C-l that was formed in the absence of heparin also undergoes spontaneous direct degradation at pH 7.5, to a complex that is similar to C-3. Additionally, this C-l dissociates very slowly to release thrombin, as demonstrated by its action on a synthetic substrate. Treatment of C-l with 1M NH2OH results in its breakdown to thrombin and antithrombin. The complex formed in the presence of heparin differs from the one formed without heparin, in that it does not exhibit any measurable dissociation and does not undergo breakdown to the C-3-type product. Moreover, whereas C-l formed in the absence of heparin is decomposed completely by 1M NH2OH, the complex formed in the presence of heparin undergoes only partial breakdown even with 2M NH2OH. Addition of heparin to C-l originally produced in the absence of heparin, has no effect on its properties.

The results thus indicate that heparin influences the mode of binding between thrombin and antithrombin as well as the rate of their interaction.
Heparin Effects (And Mechanisms) In The α2-Antithrombin Inactivation Of Serine Proteases--Thrombin, Plasmin, And Trypsin
Authors: Smith Gerald F , Sundboom Jacqueline L
Published: July 2018

It is important to elucidate the effects of heparin on the α2-antithrombin (ATIII) inactivation of serine proteases in order to understand the pharmacological activity of heparin. We have studied the enzyme kinetics of the ATIII inactivation of these proteases, and the effects of heparin on these interactions, using a common amide peptide substrate and protein substrates. We also studied the interactions of heparin with the three proteases.

We conclude that the mechanism of the catalytic effect of heparin (observed at 0. 005 unit/ml) toward the thrombin- ATIII reaction is different from the mechanism whereby heparin (only at very high concentration, e.g., 10 unit/ml) can induce an enhanced rate in the plasmin-ATIII reaction. We conclude that the first mechanism involves a heparinthrombin complex, while the mechanism with plasmin involves a heparin-ATIII complex which forms only at high heparin concentrations. This is consistent with known appropriate binding constants. We found that heparin has no effect on the very rapid inactivation of trypsin by ATIII. We further conclude that there is no common mechanism whereby clinically relevant levels of heparin cause general enhanced ATIII-protease reaction rates.

We suggest ATIII depletion during heparin therapy might be avoided by using low heparin levels, which would not allow heparin-ATIII complexes to form, yet which would catalyze the thrombin-ATIII reaction. Our finding that ATIII inactivates trypsin at a rate similar to the heparin-catalyzed thrombin-ATIII reaction suggests a physiological role for ATIII in the control of trypsin-like enzymes.
Carrier Detection Of Haemophilia A In Pregnancy By Measurement Of Factor VIIIc/Rag And VIIICAg/RAg Ratios
Authors: Mibashan R S , Peake I R , Newcombe R G , Thumpston J K , Gorer R K , Furlong R A , Rodeck C H
Published: July 2018

The 1977 WHO Memorandum on carrier detection highlighted the need to test the methods in pregnancy, when plasma modalities of facor VIII are augmented. At KCH blood was taken into citrate from 21 pregnant obligate carriers of severe haemophilia A and 28 pregnant women from unaffected families, matched for age and period of gestation. The women, who were basal after a night’s rest, were tested prior to prenatal diagnosis of haemophilia or other conditions, or to undergoing termination of pregnancy for unrelated causes.

One-stage factor VIIIC assays were done immediately; VIIIRAg (Laurell) after -40°C storage within 6 days; and VIIICAg immunoradiometrically in Cardiff on the remainder after varying intervals. Careful standardization was ensured between both laboratories.

At 17-22 weeks, VIIIRAg was elevated both in pregnant controls & carriers]; VIIIC & VIIICAg levels were also raised, though less, in the controls (values in units/dl)

In pregnant carriers, VIIIC & VIIICAg are both lower than VIIIRAg (p<0.001). Likelihood ratios of carrier probability were plotted by the unequal variances predictive method: VIIIC/RAg discriminated much better between carriers & controls than VIIICAg/RAg, due to the wider spread of VIIICAg in each group, and unlike the findings in a related study of non-pregnant haemophilia A carriers.
Specific Precipitating Antibodies To VIII: CAg In Two Patients With Haemophilia
Authors: Lavergne J M , Meyer D , Girma J M , Larrieu M J
Published: July 2018

IgG and Fab fragments were prepared from the plasma of two haemophilia A patients whose anti- V111 : C titres were 2,000 and 400 Bethesda U./ml. 125-I-IgG and Fab specific for VIII :CAg were purified by a solid phase procedure and the reactivity of these two antibodies with VIII: CAg was studied by immunoprecipitation in agarose. With double diffusion and autoradiographic methods, both the IgG and Fab antibodies showed a precipitin line against normal plasma, serum, haemophilia A+ plasma, cryopre ci pi t ate , purified F.VIII/vWF or VIII: C free of vWF. No precipitin line was observed in haemophilia A- or severe von Willebrand’s disease. With electroimmunoassay (radi o-Laurell) using both IgG and Fab antibodies, results of VIII: C A g were in agreement in all samples with those obtained by immunoradiometric assay using the same antibodies. The specificity of the immunoprecipitation observed in agarose with IgG or Fab fragments was assessed by modifying the pH (7.5 to 9.5) of the buffer, the ionic composition (0.15, 0.5 and 1M sodium chloride, 0.15 and 0.5M potassium iodide, 0.15 and 0.5M sodium thiocyanate) of the washing fluid,or by carbamy1ation of the anti-VIII: CAg IgG. In all cases, specific precipitation was observed. In addition, 125-I-IgG or Fab isolated by the same technique from normal plasma gave no precipitinline with VIII: CAg. This study demonstrates that, contrary to previous evidence, 1) human anti-VIII: CAg antibodies are both precipitating as well as neutralizing when studied by highly sensitive techniques; and 2) monovalent Fab can also precipitate VIII: C Ag. The results of this study raise some questions about the lattice theory of immunoprecipitation.
Assessment Of Venous Function After Major Gynecological Surgery With Heparin-Dihydroergotamine In The Prophylaxis Of DVT
Authors: Kunz S , Burth O M , Zimmerer E
Published: July 2018

Dihydroergotamine (DHE) has been shown to have synergistic effects with heparin in the prophylaxis of DVT. To assess the hemodynamic efficacy of DHE in clinical conditions, 72 patients undergoing hysterectomy were randomly allocated to three groups receiving heparin/DHE, heparin or acenocoumarol. Venous function was measured by occlusion plethysmography and doppler ultrasonography. Diagnosis of DVT by 125-I-fibrinogen test.

Results: Doppler Ultrasonography: The velocity of venous flow in the left V. femoralis was lower than at the right side in all groups before hysterectomy. After surgery, a significant increase from 11.2 to 15.5 cm/sec in the heparin/DHE group could be demonstrated. Heparin and acenocoumarol patients showed a tendency to lower venous return. The differences were more marked in women with vaginal hysterectomy.

Occlusion plethysmography: No differences in venous capacity before and after operation could be found in the heparin and acenocoumarol group. A significant reduction (16%) was measured in heparin/DHE treated patients. The maximal venous outflow also was significantly reduced in this group (18%). This difference also was found in patients with varicose veins.

Conclusions: The data confirm that DHE increases the velocity of venous blood flow in the legs by constricting the capacitance vessels. This haemodynamic effect could be demonstrated in clinical conditions.
Extracranial Carotid Artery Disease Assessed By Indium-111 Labelled Platelets In Man
Authors: Goldman M , Simpson D , Hawker R J , Drolc Z , McCollum C N
Published: July 2018

Platelet microemboli have been implicated in the aetiology of transient ischaemic attacks and strokes. Current non-invasive diagnostic methods are restricted to assessing blood flow. Arteriography may demonstrate non-occluding atheromatous ulcers but carries a significant morbidity.111-In labelled platelets and gamma camera imaging of diseased carotid vessels have therefore been evaluated.

Eight patients proceeding to unilateral carotid endarterectomy received an injection of autologous platelets labelled with 111-In-oxine 48 hours preoperatively. Gamma camera images were taken at 4 and 24 hours. Operative endarterectomy specimens were collected and gamma emissions were measured.

The scintigraphic images were independently assessed by two observers. Nine abnormal vessels were identified by platelet accumulation. In 5 cases the observers combined assessment was in complete accord with previous bilateral angiography. Individually each observer identified the symptomatic side in 6 patients. Activity in the endarterectomy specimens measured ex vivo varied from 1.22-84.5 times greater than background activity. The ratio of specimen activity to 5 ml blood samples simultaneously taken varied from 0.017 to 0.360 (mean 0.089).

This non-invasive technique allows localisation of platelet activity in the extracranial cerebral circulation. Objective assessment in vivo is hindered by the relatively small amount of activity on the diseased vessels by comparison with circulating blood activity.
Arachidonate-Induced Fibrinogen Binding To Thrombin-Degranulated Rabbit Platelets Is Independent Of Released ADP
Authors: Harfenist E J , Guccione M A , Packham M A , Kinlough-Rathbone R L , Mustar J F
Published: July 2018

When human or rabbit platelets are stimulated with ADP, fibrinogen (Fbg) binding sites are revealed, the platelets bind Fbg and aggregate. Since stimulation with other aggregating agents (arachidonate, collagen or ionophores) releases platelet granule contents, including ADP and Fbg, it is difficult to determine whether these agents cause aggregation or Fbg binding that is independent of ADP. Therefore we treated rabbit platelets with thrombin (0.73 U/ml) to release at least 90% of their dense granule contents, as measured by 14C-serotonin, washed and resuspended them, and studied aggregation and Fbg binding upon stimulation with ADP or arachidonate. In the presence of Fbg, thrombin-degranulated platelets (TDP) aggregate in response to ADP or arachidonate at concentrations that aggregate untreated platelets, although TDP aggregate somewhat less extensively. When TDP are aggregated with 50 μM arachidonate, they lose up to 9% of their remaining serotonin, corresponding to a concentration of ADP in the suspending medium of not more than0.06μM, which does not aggregate TDP or cause detectable Fbg binding. When creatine phosphate/creatine phosphokinase (CP/CPK) is added at a concentration that abolishes aggregation in response to 1 μM ADP, it reduces aggregation caused by arachidonate by only 18%. Binding studies with 125I-Fbg show that stimulation of TDP with either ADP or arachidonate results in specific Fbg-binding similar to the binding to ADP-stimulated normal platelets. CP/CPK almost completely inhibits binding induced by ADP but reduces binding induced by arachidonate by only 30%. Aggregation and binding studies with TDP using a combination of arachidonate with low concentrations of ADP failed to show synergistic effects. Thus arachidonate causes aggregation and Fbg binding to TDP that are independent of ADP, although the magnitude of these effects may be increased by released ADP.
Role Of Prostaglandins In Platelet Fibrinogen Receptor Exposure
Authors: Bennett J S , Vilaire G , Burch J W
Published: July 2018

Fibrinogen binding to membrane receptors exposed by agonists such as ADP and thrombin is a prerequisite for platelet aggregation. However, a role for platelet prostaglandins in this process remains to be clarified. To assess whether platelet prostaglandins can expose fibrinogen receptors, we examined the effects of aspirin (ASA) and indomethacin on fibrinogen binding to ADP and epinephrine- stimulated platelets. Fibrinogen binding was measured by incubating gel-filtered human platelets (GFP) with 125I- labeled fibrinogen, CaCl2 and ADP or epinephrine at 37°C for 3 min without stirring. Free and platelet-bound I-fibrinogen were separated by rapid sedimentation of the platelets through silicone oil. The platelet release reaction, measured as 14C-serotonin secretion, did not occur under these conditions. Fibrinogen binding in response to l-2μM ADP or 10μM epinephrine was inhibited 40-60% by preincubation of platelet-rich plasma with 1 mM ASA for 20 min at 37° C. Similar results were produced by adding 25μM indomethacin to GFP. The inhibitory effect of ASA on ADP-stimulated fibrinogen binding could be overcome by increasing the ADP concentration while the inhibition of epinephrine-stimulated fibrinogen binding could not. However, stimulation of aspirin-treated platelets with both 10μM epinephrine and the prostaglandin endoperoxide PGH2(1μM) restored the extent of fibrinogen binding to that of control platelets stimulated by 10μM epinephrine alone. Scatchard analysis demonstrated that ASA decreased the number but not the affinity of the exposed fibrinogen binding sites. Identical results were obtained if ASA was ingested before blood donation. These studies demonstrate that prostaglandins synthesized in response to platelet stimulation can expose fibrinogen receptors on the platelet surface. Furthermore, these studies support the concept that platelet aggregation can occur through mechanisms independent of platelet ADP secretion.
Fibrinogen (FIBR) Binding Is Necessary For Incorporation Of 125I-Labeled Membrane Protein Into The Platelet Cytoskeleton
Authors: Zucker M B , Grant R A
Published: July 2018

Platelets stimulated with ADP bind fibr specifically, and this ability correlates well with their ability to aggregate when they are shaken with ADP and fibr. Others found that platelets incubated with chymotrypsin (CT) bind and aggregate with fibr without stimulation by ADP; as with ADP-induced stimulation, these responses are inhibited by excess EDTA. Phillips et al. showed that thrombin activation produced a large Triton-insoluble cytoskeleton in the presence of either EDTA or CaCl2, but that 125I-labeled glycoprotein(s) IIb-III were incorporated into the cytoskeleton only when CaCl2 was present and aggregation occurred. This system cannot be used to assess the role of fibr in membrane glycoprotein incorporation, as exogenous fibr is not required for thrombin-induced aggregation. We therefore used CT-treated platelets labeled with 125I. They were suspended in calcium-poor Hepes-buffered Tyrode’s solution at 22°C for 30 min with 2.5 mM EDTA or 1 mM CaCl2 with or without 100 ug/ml fibr. Only samples with CaCl2 + fibr aggregated, and similar samples prepared with CT-treated platelets which had first been incubated with EDTA at 37°C for 15 min failed to aggregate despite recalcification. Cytoskeletons were prepared by adding an equal vol of 2% Triton X-100-5 mM EGTA-0.1 M Tris pH 7.4 and centrifuging 1 hr later. Cytoskeletal protein was 10-25% of total and did not vary with aggregability. CT-treated platelets showed more 125I bands than untreated labeled platelets. The two bands with highest Mr on SDS-PAGE were present only when the platelets aggregated during incubation, i.e., under conditions in which fibr is bound. Possibly the dimeric fibr molecule must link two surface molecules before they can attach to the cytoskeleton.