bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of 5HT Uptake by Picryl Sulfonic Acid Amino Group Modification in the Platelet Membrane
Authors: Harbury C.B. , Janszen M.E. , Rutherford S.
Published: May 2019

The main purpose of this experiment was to assess the functional importance of platelet membrane amino groups (NH2). Picryl Sulfonic Acid (PSA) is a known NH2 modifier. Platelets were harvested, exposed to 0.25 - 32 mM PSA for 15 minutes at 22°C. They were then washed and functionally tested. The release was measured by C5HT release. The release reaction was induced by 0.1, 1, and 10 u/ml Thrombin (THR), Trypsin (TRY), Collagen (COL), and Latex particles (LAT). There was gradual but never complete inhibition of the release reaction. At 16 mM PSA, the platelets started to lose small amounts of 5HT (4%). Clot retraction was not affectedby 1 to 32 mil PSA. 5HT uptake was enhanced at 0.1 to 4 mM PSA (paired t test, p<0.01) and maximal at 2 mM PSA.Pre-incubation with 0.25 and 0.5 mM PSA in the presence of calcium enhanced the PSA effect on rate of 5HT uptake (paired t test, p<0.01). This may be due to the enhanced availability of amino groups to PSA in the presence of Ca, speculated by Godin to be due to a lipid effect (1972). Incorporation of PSA into the membrane was increased in the presence of Ca (O.D. at 335 nm). In summary, PSA modification of platelet membrane NH2 groups partially but never completely interferes with the induction of the release reaction. The inhibition was not specific with respect to the release inducing agent. Clot retraction remains intact. The observed significant enhancement in rate of 5HT uptake may be specific or may be due to neutralization of the charge on free amino groups, which repels 5HT.
Leucocyte Adherence of Arterial Endothelium
Authors: Mayrovitz H.N. , Wiedeman M.P. , Turna R.F.
Published: May 2019

Adherence of leucocytes to the endothelium of microvessels is one of the initial responses seen after tissue injury. Leucocyte adherence and their occasional emmigration into adjacent tissue is considered to mark the beginning of an inflammatory process. The underlying mechanisms responsible for the initial adherence is unknown. A method of studying this problem in vivo is complicated by the fact that most experimental preparations to expose the microvessels for direct microscopic observation involve surgery which itself is sufficient to promote adherence. In the present studies, laser irradiation of microregions of tissue at various distances from arterioles in the intact bat wing were used to probe the time course of trauma-induced changes in leucoctye-vessel wall interaction. It was found that, for equivalent tissue injury, the time required for normally nonadherent leucocytes to begin sticking and rolling along the arterial wall was dependent on the square of the distance between tissue injury site and the vessel wall. At a fixed distance, the latency time decreased by an amount which was inverserelated to the extent of tissue injury. The measured latency times, which ranged from 15 to 400 seconds, shows that the primary mechanism causing leucocyte adherence may act much more rapidly than previously thought. Calculations indicate that the measured latency times may be accounted for either by a laser released tissue substance having a diffusion coefficient of 5 × 10-7, or possibly due to temperature elevation of the vessel wall by heat from the laser beam.
Effects of Lectins on Blood Platelets from Various Species
Authors: Tangen O. , Karlstam B. , Bygdeman S.
Published: May 2019

Earlier it has been shown that different lectins induce a variable degree of aggregation of platelets. The present study confirmed previous data and demonstrated that wheat germ agglutinin (WGA) was very active, 1eucoagglutinin had about a tenth of the activity of WGA on a concentration basis, and Con A had a weak aggregating effect on human gel filtered platelets (GFP). Soy bean lectin did not aggregate human GFP.

The fact that adenosine inhibited WGA- and leucoagglutinin-induced aggregation that WGA and Con A caused serotonin release, and that the aggregation- curves indicated platelet shape change are indications that the lectins influenced glycosyl moieties involving one or more molecules relevant to release and aggregation reaction.

GFP were markedly more responsive to the lectins than platelets in plasma, probably due to interfering glycosyl groups amongst the plasma constituents.

Platelets from man, rabbit, rat, cow and pig reacted differently towards the lectins, human platelets being the most reactive and bovine and porcine platelets being almost unreactive. These results pose intriguing questions regarding the glycosyl content of platelet membranes in different species and their relation to platelet release and aggregation.
Effects of Varying Concentrations of Dietary Linoleic Acid on Platelet-Rich Plasma Clotting Time, Platelet Aggregation and Phospholipid Fatty Acids Composition of Rats
Authors: McGregor L. , Renaud S.
Published: May 2019

A high intake of saturated fats has been shown, in rats fed drastic diets, to be associated with an acceleration of platelet-rich plasma clotting time, an increased thrombin aggregation and a high incidence of venous thrombosis. Linoleic acid appeared to have an antithrombotic effect. In order to understand the role of dietary linoleic acid in platelet function, the present study was undertaken with different concentrations of linoleic acid (0%, 0.5%, 1% and 6%), provided by corn oil which were added to purified diet high in saturated fat (40% hydro-genated coconut oil) but otherwise completely adequate in any other aspects. These four diets were fed to 4 groups of weanling male Sprague-Dawley rats for 4 months. At the end of the feeding period, platelet-rich plasma clotting time, platelet aggregation to thrombin, ADP, arachidonate, collagen and fatty acid composition of total platelet phospholipids were determined. Rats fed 0%, 0.5%, 1% linoleic acid showed a marked hypercoagulability, hyperagregability to thrombin, in comparison to 6% linoleic acid-fed rats. Moreover, this hyperagregability and hypercoagulability were associated with the following increased ratios of platelet phospholipid fatty acids : 20 : 4/18 : 2 and 16 : 1 + 18 : 1 + 20 : 1 + 24: 1 + 20 : 3ω9/18 : 2. It seems that dietary linoleic acid plays a crucial role in platelet functions.
Effect of Warfarin, Warfarin Enantiomers and Defibrase on Lewis Lung Carcinoma Metastasis Growth in Mice
Authors: Donati M.B. , Poggi A. , Mussoni L. , Kornblihtt L. , Gaetano G.de , Garattini S.
Published: May 2019

The Lewis Lung Carcinoma (3LL) is a spontaneously metastasizing tumor, the development of which is eccompanied by marked hemostatic changes. Acceleration of labelled fibrinogen turnover and fibrin accumulation at the tumor and metastasis sites have been observed in 3LL bearing mice. Both the number and the weight of lung metastases were significantly lower in mice anti-coagulated with racemic sodium warfarin during the whole tumor development period (prothrombin complex activity around 20%). This treatment had less effect on the primary tumor. The anti-metastatic effect was slightly greater with larger doses of warfarin, which lowered the prothrombin complex activity to less than 5%. Continuous anticoagulation also protected the animals from pulmonary growth of blood-borne tumor emboli following intravenous injection of 3LL cells. This effect appeared to be closely associated with the anticoagulant activity of warfarin; indeed experiments performed with the resolved warfarin enantiomers showed that R-warfarin had virtually no anticlotting activity in mice and did not modify the metastatic growth of 3LL cells; the opposite was true for S-warfarin.

Lung metastasis growth was increased in mice kept defibrinogenated during the whole period of tumor development by treatment with batrcxobin; in contrast, in mice kept defibrinated only during the period of metastasis growth, with or without surgical removal of the primary tumor, metastasis formation was slightly decreased. This suggests that fibrin may play different roles in various phases of metastaticspreading of the same tumor.
Effect of Bezafibrate on Blood Coagulation and Drug Interaction with Phenpro-Coumon.
Authors: Zimmermann R. , Hoffrichter A. , Walter E. , Lang P.D. , Ehlers W. , Andrassy K. , Schlierf G. , Weber E.
Published: May 2019

In addition to their effect on lipids Clofibrate and related drugs can influence platelet function and fibrinolytic activity. Interaction of these drugs with coumarin anticoagulants may induce hemorrhagic complications. Therefore the effect of a new hypolipemic agent (bezafibrate) on blood coagulation and components of the fibrinolytic encyme system was investigated.15 patients on long term treatment with phenprocoumon received bezafibrate (450 or 600 mg daily) for 4 weeks. Evaluation of platelet function demonstrated a reduced platelet aggregation induced by collagen (p < 0.05) and prolongation of bleeding time (p< 0.05), dependent on the given dose. Plasma fibrinogen was reduced moderately. Examination of the fibrinolytic activity yealded a shortened euglobulin clot lysis time (p < 0.05) but no changing of inhibitors. Serum level of phenprocoumon and bezafibrate were determined to obtain data about the mechanism of drug interaction. After reduction of the phenprocoumon dose by 20.6 or 28.7% the serum phenprocoumon levels decreased by 9-3 and 30.6%, respectively.Hypo-prothrombinemia was maintained (p< 0.05). The results support the hypothesis, that hypolipemic drugs such as bezafibrate augment the anticoagulant response to phenprocoumon by increasing the affinity of the receptor site for coumarin and not by effecting the rate of metabolism.
Hematologic and Coagulation Measurements in Autotransfused Blood
Authors: McKenzie F.N. , Wall W. , Heimbecker R.O. , Barr R. , Robert A.
Published: May 2019

Reinfusion of blood shed during elective or emergency vascular surgery (autotransfusion) is an under utilized technique. This is due in part to doubts as to the quality of autotransfused blood and concern about the risk of inducing a coagulopathy in the recipient. We have measured coagulation and hematologic parameters in autotransfused blood in the recipient before and at intervals after operation in 62 patients, none of whom received bank blood or blood products at the time of the study. The patients were heparinized (3 mg/Kg) during operation and this was reversed by protamine at the end of the procedure. The salvaged blood was reinfused immediately after appropriate samples had been taken. The mean volume of blood autotransfused was 1.8L in 58 patients and 9.4L in 4 patients. There was excellent preservation of platelets and fibrinogen, normal levels being maintained both in the autotransfused blood and in the recipients. Values for fibrin degradation products and euglobulin clot lysis remained normal. The mean plasma hemoglobin in the autotransfused blood was 416 mg% and this was not correlated to the volume autotransfused. Partial thromboplastin time which was prolonged by heparin during surgery was consistently normal post-operatively. No patient developed complications which could be attributed to autotransfusion and, in particular, re-operation for post-operative bleeding was never required and wound hematoma was not seen. We conclude that autologous blood may be returned to patients in large amounts without significant risk using the technique described. The technique deserves wider application in major surgery.
Differential Determination of Antithrombin Activities of Plasma Antithrombin III and α2-Macroglobulin
Authors: Matsuda T. , Ogawara M. , Hirabayashi N. , Seki T. , Murakami M.
Published: May 2019

Among several thrombin inhibitors in normal plasma, antithrombin III (AT-III) and α2-macroglobulin (α2-M) are especially important.However, there has been no appropriate method to determine AT-III and α2-M separately. This study was carried out to differentiate the actions of these two inhibitors of thrombin in plasma. By using single radial immunodiffusion method, we observed that AT-III was adsorbed to bentonite, although α2-M was not. It was necessary to incubate 1 ml of oxalated plasma with 300 mg of bentonite at 37°C for 10 minutes to remove AT-III completely. Supernated plasma contains neither AT-III nor fibrinogen which affects antithrombin activity of plasma. From these results, it is evident that antithrombin activity of plasma adsorbed with bentonite was attributed to that of α2-M. Antithrombin activities of plasma adsorbed with bentonite in 20 healthy subjects were significantly correlated with concentrations of α2-M in plasma, measured immunologically (r=+0.87, p<0.001). It is reasonable to presume that difference between antithrombin activity of defibrinated plasma by heating at 56°C for 2 minutes and that of bentonite treated plasma mainly indicates activity of AT-III. These differences measured in 20 healthy individuals were significantly correlated with plasma AT-TH levels, assayed immunochemically (r=+0.53, p<0.01).
Aspirin-Persantin Prophylaxis in Elective total hip Replacement
Authors: Silvergleid A.J. , Bernstein R. , Burton D.S. , Tanner J.B. , Silverman J.F. , Schrier S.L.
Published: May 2019

A prospective, double-blind clinical study was performed to evaluate the combination of dipyridamole(Persantin)225 mg/day and acetyl salicyclic acid (ASA) 1 g/day prophylaxis of post-operative venous thromboembolism in elective total hip replacement. Patients were stratified according to age, and randomly assigned to receive drug or placebo. All patients were followed with 125I-labelledfibrinogen scanning for one week post-operatively, or until fully mobile. Venography was performed in 67/129 patients; in 27 patients the venogram was obtained to confirm a positive fibrinogen scan, in 40 patients an elective venogram was obtained on the 7th post-operative day to evaluate the operated thigh (a blind area for scanning). Thrombosis (by scan or venogram) was found in 16/66(24%) in the control group, and in 21/63(33%) in the treated group. Overall incidence was 37/129 (29%). Correlation of scan with venography was 90%. There were no clinically significant pulmonary emboli in either group. We conclude that the combination of ASA and dipridamole as given in this study is not effective prophylaxis in elective total hip replacement.
Diagnosis of the Prethrombotic State. Introduction
Authors: Sixma Jan J.
Published: May 2019

The purpose of this symposium is to highlight new developments in methods that may detect patients with increased risk for arterial or venous thrombosis. Some of the techniques presented rely on the presence of changed clotting factors, fibrinogen in particular, or of released peptides. A novel approach is the use of antibodies directed specifically against complexes of inhibitors with activated factors.

Blood platelets play an important role particularly in arterial thrombosis.Radio-immuno-assays have been worked out for two secretion products from platelets: platelet factor 4 and beta-thromboglobulin. Theoretically the sensitivity of these tests will be limited by the short half life of the substances. A possible useful approach is therefore the study of the properties of released platelets since it has been demonstrated that these platelets may have a normal survival. The platelet coagulant activity as predictor of thrombosis may fit in here. Other properties of released platelets such as the exposure of actin or the decreased uptake of serotonin are currently under investigation. Spontaneous aggregation in vivo or in vitro as well as a short half life of labeled platelets has been found in various thromboembolic diseases.

The predictive value of various tests should be evaluated in prospective studies. An example of such an approach is given in the paper that concludes the symposium.
Platelet Survival Measurement - Clinical Results.
Authors: Genton E.
Published: May 2019

Platelet survival (PS) time has been studied in patients in a variety of clinical circumstances associated with abnormal numbers of platelets, suspected thrombotic process, or after platelet suppressing drug administration.

In conditions with thrombocytopenia, PS may differentiate decreased platelet production (ineffective thrombopoiesis or megakaryocyte hypoplasia) where PS is normal from increased destruction of platelets where PS is shortened. Increased destruction may arise from: extrinsic mechanisms, e.g. immunologic (ITP-SLE-drug reactions) or abnormal surface (diseased endothelium or foreign surface) are associated with short PS, often to extreme degrees; intrinsic platelet defect (Wiskott-Aldrich Syndrome), where autologous PS is shortened and isologous PS is normal.

In thrombotic disorders, PS is shortened during active thrombosis and may be chronically shortened in conditions with recurrent thrombosis (homocystinemia, atheroslcerosis, valvular heart disease). The test may prove useful in prognosis (e.g. valvular heart disease patients with shortened platelet survival may have higher risk of embolic events than with normal PS).

Only a few platelet suppressing drugs (including sulfinpyrazone and dipyridamole) affect a shortened platelet survival time. These reduce thrombosis in patients with prosthetic heart valves and silastic AV shunts, suggesting PS may identify useful drugs.

Thus, PS may differentiate disease conditions associated with abnormal platelets, predict the course of patients at high risk of thrombotic complications, and identify clinically useful platelet suppressing drugs, or serve as a monitor for measuring effects of drug treatment.
Haemostatic Findings in Men with and without Ischaemic Heart Disease.
Authors: Meade T.W. , Chakrabarti R. , Haines A.P. , North W.R.S. , Stirling Y.
Published: May 2019

The main purposes of the prospective Northwick Park Heart Study (NPHS) are to improve the prediction of arterial disease, especially ischaemic heart disease (IHD), and to elucidate its pathogenesis. Some 1800 men and 800 women have so far been recruited. Measures of haemostatic function are made along with those of variables (E.G. cholesterol) already known to be associated with IHD;all participants are followed up. Of the first 873 white men aged 40-64 at entry, 18 had already had a “definite” myocardial infarction (MI) at least 2 years previously, and 18 had “definite” angina pectoris (AP). Recruitment values for factors II, V, VII, VIII and X, fibrinogen, antithrombin III, α2-macroglobulin, fibrinolytic activity, platelet count and platelet adhesiveness are compared with similar values in those free of clinically manifest IHD and with normal electrocardiograms (ECGs). Those with MI had significantly higher levels of fibrinogen, and platelet adhesiveness, and significantly lower fibrinolytic activity than those without IHD and normal ECGs;their factor VII and VIII levels were also higher. Changes in those with AP were similar, though not as great as in those with MI. There were no differences in those without clinical manifestations of IHD but with ischaemic ECGs. IHD prevalence data, based on results after the clinical event, generally give less striking contrasts than incidence data (which NPHS will eventually provide), in which recruitment findings are related to the subsequent onset of disease.Nevertheless, the prevalence findings are compatible with the concept of a “hypercoagulable state” in which thrombosis is the main complication of atheroma, and responsible for many of the clinical manifestations of IHD.
Endothelium and Intimal Thickening
Authors: Stemerman M.B.
Published: May 2019

A major hypothesis of atherogenesis ascribes endothelial damage as a causation of intimai proliferation which may, in turn, be a progenitor of arteriosclerosis. This hypothesis appears substantiated by experimental evidence varying from immunologic to mechanical damage of endothelium as a means of inducing both diffuse and focal intimai proliferation. There appears, in addition, other influences exerted by endothelial cells on the pathophysiology of intimai thickening. During intimai healing after wide spread endothelial denudation by an intraarterial balloon catheter, endothelial regrowth seems to promote regression of intimai thickening. It is not clear if this diminution of thickness is due to reduction in cells, extracellular material, or both.

However, diminution of intimai thickening is not a feature of the healing process in the presence of hyperlipidemia. With widespread de-endothelialization, as described above, areas beneath regenerating endothelium are much thicker, and show a marked predisposition for the accumulation of lipid into the underlying already thickened intima. What physiologic or metabolic influences are exerted by the endothelium under these circumstances are unclear, but, the presence of endothelium may be a highly significant factor in the natural history of intimai proliferation and atherogenesis.
Synthesis of Procollagen by Endothelial and Smooth Muscle Cells in Vitro
Authors: Macarak E. , Kefalides N.A.
Published: May 2019

The synthesis of procollagen by either calf endothelial cells or smooth muscle cells in culture has been studied. Endothelial cells were characterized by the presence of factor VIII antigen and Weible-Palade bodies. Endothelial or smooth muscle cells incorporated [14C]proline into non-dialyzable protein and synthesized hydroxy[14C]proline. Gel filtration (Agarose A5m-SDS) indicated major differences in the molecular properties of the [14C]procollagen and [14C]-non-collagen proteins synthesized by these cells. Endothelial cells synthesize a procollagen component which, after reduction, contains approximately 50% of the hydroxy[14C]proline synthesized and elutes near the exclusion volume. This fraction contains 14% of the total hydroxylase] proline as the 3-isomer. Two additional components eluted with apparent mol. wts. of 250,000 and 140,000. Only the 140,000 mol. wt. component contains hydroxy[14C]proline. Gel filtration (Agarose A5m-SDS) of the [14c]protein synthesized by smooth muscle cells showed only a single component eluting with an apparent mol. wt. of 130,000. The percent hydroxyla-tion of proline in the peak fraction was 44. The percent of 3-hydroxy [14C]proline was 2%. These data indicate that endothelial cells and smooth muscle cells in culture most likely synthesize different kinds of procollagen (Supported by USPHS Grant AM-14526, HL-18827 and HL-15061).
The Evolution of Vertebrate Fibrinogen
Authors: Doolittle Russell F.
Published: May 2019

The fibrinogens of all vertebrates studied to date are comprised of three pairs of non-identical polypeptide chains.Moreover, in all of these cases--whether the molecule is isolated from a primitive fish or an advanced mammal--polymerization is induced by the thrombin-cata-lyzed removal of polar peptide material from the ami no-terminal segments of two of the three chains. Amino acid sequence studies on mammalian fibrinogen molecules have provided convincing support, however, for the notion that the three non-identical polypeptide chains have evolved from a common ancestor and also that the present chains have been elongated by a series of internal duplications.Thus, we are now in a position to speculate on the structure of the primordial fibrinogen molecule and how it could be gelled by a proteolytic event. The molecule must have been comprised of (two) bundles of three identical polypeptide chains, all of which must have been sensitive to the action of thrombin.Furthermore, symmetry arguments suggest that the way the three identical chains would have been bundled together is best approximated by a (disulfide-linked) three stranded rod. Given the multiple restraints on such a molecule, a reasonable scheme can be advanced for polymerization based on the removal of polar material from one end of the rod. The differences in amino acid sequences for the three chains comprising the present day fibrinogen molecules are such that the duplicative events leading to their divergence must have occurred more than 600 million years ago, or long before the origin of vertebrates. It will be of great interest if a primitive molecule can be found in some extant creature which approximates the prototype structure.
The Control of Platelet Function by Cyclic amp and Thromboxane Synthesis
Authors: Bang N.U. , Boxer M.G.J. , Heidenreich R.O. , Ho P.P.K. , Schmidt M.J.
Published: May 2019

Platelet (P) levels of cyclic AMP (cAMP) and thromboxane (TX) synthesis have been identified as major regulators of P aggregation and release. We have utilized as probes drugs which either decrease TX synthesis by cyclooxygenase inhibition (aspirin and indomethacin) or which increase P cAMP (adenosine; PGE1; theophylline; isobutyl methyl xanthine; and SH-869, a dipyridamole analog) to evaluate relative contributions of cAMP and TX and their possible interreactions in mediating P function. Cyclooxygenase inhibitors at concentrations 8-16 fold lower than those inhibiting P aggregation and release caused almost complete inhibition of TXB2 synthesis from exogenous 14C-arachidonic acid (aa) and malonyldialdehyde (MDA) production in P stimulated by thrombin (T) or N-ethylmaleimide (NEM). Drugs elevating P cAMP at concentrations equal to or greater than those causing complete inhibition of P aggregation and release did not inhibit TXB2 synthesis from exogenous 14C-aa, nor did they inhibit MDA production in P stimulated by NEM or by concentrations of T sufficient to produce maximal TX synthesis.However, these drugs variably inhibited MDA production when P were stimulated at lower T concentrations causing only minor TX synthesis.Thus, elevated P cAMP did not inhibit TX synthesis from aa but appeared to weakly inhibit aa phospholipase. We conclude that TX synthesis cannot be the sole, final mediator of P aggregation and release but that these events result from as yet unidentified mechanisms modulated largely independently by TX synthesis and intra-P cAMP levels.
Aspirin, Collagen and the Collagen-Platelet Interaction
Authors: Whitin John C. , Simons Elizabeth R.
Published: May 2019

While the effect of aspirin (ASA) on platelets has been investigated by many laboratories, the effect of ASA on collagen in collagen-initiated platelet aggregation has not. We report here on the rate of collagen fibril formation in the presence of ASA from 0.1 to 15 mM, and on the resultant perturbation of platelet aggregation by such ASA-treated collagen. The kinetics of fibril formation of rat tail tendon collagen are linear at 30 C in the absence of perturbants as well as in the presence of low (<7.5mM) concentrations of ASA. Higher (>7.5mM) concentrations of ASA lead to an inductive period whose length is a function of ASA concentration. The rate of fibril formation increases gradually, eventually attaining the control level. Fibrils formed in the presence of lOmM ASA contain one 14C acetyl group per collagen α chain. Parallel experiments with sodium salicylate (NaSA), sodium acetate (NaAc) and acetyl imidazole (AI) were also performed. NaSA and NaAc did not perturb collagen fibril formation. AI, on the other hand, closely resembled ASA in perturbation of fibril formation and of platelet aggregation. The effect on platelets of aliquots of equal size fibrils (as monitored by light scattering) can be compared. Collagen fibrils formed in the presence of ASA or AI do initiate a platelet response even though collagen NH2 groups have been acetylated. At high ASA, AI, or NaSA concentrations these measurements were perturbed by the large amount of residual unreac-ted reagent in the fibril aliquots. These in vitro results indicate that aspirin treatment slows but does not prevent the formation of collagen fibrils capable of interacting with platelets. The effects may be significant, particularly for collagen newly synthesized in vivo in the process of repair.
Rapid Isolation and Purification of Antibodies to Coagulation Factors by Protein A
Authors: Lee H.H. , Tucker D. , Allain J.P.
Published: May 2019

A simple and reproducible method has been developed for rapidly isolating and purifying antibodies to Factor VIII from the plasma of both haemophilic and non-haemophilic patients. The one-step separation of these immunoglobulins makes use of the high affinity binding of staphylococcal cell wall protein A with the Fc region of the human γG1, 2 and 4 subclasses but not with the γG3 subclass. Plasma, as little as 0.5 ml, can be chromatographed on protein A Sepharose CL-4B gel columns. The majority of the plasma proteins, including IgG3, are not bound to the protein A, and are eluted as the first peak at neutral pH. Bound proteins are eluted as a second peak at pH 2.4. Plasmas from ten haemophilic and ten non-haemophilic patients with antibody to Factor VIII have been fractionated by this method. Without exception, the anti-factor VIII activity was bound to the protein A gel and thus eluted in the second peak. The yield of antibody was 70-80%. It is concluded that the great majority, if not all, of the antibodies to factor VIII belong to one or more of the IgG subclasses 1, 2 and 4 and can therefore be efficiently isolated by protein A chromatography. The plasma of a patient with antibody to factor V was also fractionated by protein A gel with similar results.
Platelet Lifespan in Pregnancy Complicated by Pre-Eclampsia
Authors: Schieppati A. , Cossu M. M. , Rossi E. , Remuzzi G.
Published: May 2019

Increased platelet consumption has been suggested to occur in women who develop pre-eclampsia and to play a pathogenetic role in this disorder. The approach to this problem was difficult until a non radioisotopic technique was developed to measure platelet lifespan (Stuart et al. , NEJM 1975, 292, 1310). Malondiafdehyde (MDA) is measured in platelet rich plasma (challenged with thrombin) before and at intervals after ingestion of 500 mg aspirin. The time taken for MDA values to return to baseline levels after aspirin is equal to platelet lifespan. A study is in progress to compare platelet lifespan in healthy non pregnant women, in women in the third trimester of uncomplicated pregnancy and in patients with pre-eclamps. The results obtained so far suggest that platelet lifespan in pregnancy, either normal or complicated with mild pre-eclampsia, is within the normal range. Indeed platelet lifespan was 7.6 , 9.6, 10.5, and 9.5 days in 4 women with uncomplicated pregnancy and , 10.0, 11.7, and 9.0 days In 3 patients with mild pre-eclampsia. Platel ellifespan in non pregnant women ranged between 9.4 and 12.2 days.
Gel Filtration of Plasma from Rabbits Injected with Honomeric DES-AB 125I-Fibrin and 131I–Fibrinogen
Authors: Kröhnke I. , Hahn I. , Krell W. , Müller-Berghaus G.
Published: May 2019

We intended to study the chromatographic behaviour of soluble des-AB fibrin prepared in vitro and injected into rabbits. To prepare des-AB 1251-fibrin, purified rabbit 125I-fibrinogen was clotted by thrombin and the formed clot dissolved in Tris-buffered 3 M urea. Gel filtration of des-AB fibrin in urea through sepharose-CL 6B columns equilibrated with buffered 3 M. urea revealed monomeric fibrin. Rabbits received 131I-fibrinogen and 5 min later monomeric des-AB 125I-fibrin in urea. 30 min after injection blood was drawn and the plasma obtained filtered through sepharose-CL 6B columns eguilibrated with buffered plasma. At 20°C as well as at 37°C des-AB 125I-fibrin was eluted in the void volume in front of the 131I-fibrinogen peak. The data demonstrate that monomeric des-AB 125I-fibrin in urea injected into rabbits remains soluble in plasma. Possibly, monomerJ fibrin is converted to circulating soluble high-molecular weight fibrin aggregates or fibrin monomer changes its molecular configuration, thus showing a different gel filtration behaviour.

(Supported by the Deutsche Forschungsgemeinschaft).
Tissue Localization of Fibrin Degradation Products.
Authors: Emeis J.J. , Nieuwenhuizen W. , Lindeman J.
Published: May 2019

Tissue distribution of fibrin degradation products (FOP’s) during disseminated intravascular coagulation (DIC) is still largely unknown, since present immunohistochemical methods cannot discriminate between FDP’s and other fibrin-related material. Using tissue from rats with thrombin-induced DIC, we developed an immunoenzymehistochernical method for specific FDP-localization. This method is based on the following observations:

1. FDP’s are - in contrast to fibrin - retained in ethanol-fixed tissue only; however, not in formaldehyde-fixed tissue;

2, FDP’s in tissue will react with some of the antisera employed only (antisera against rat fibrinogen, FDP-D and E, and the constituent chains of fibrinogen were tested), whereas fibrinogen and fibrin will react with all said antisera.

At 20-30 min after thrombin injection, FDP’s were found in the epithelial cells of kidney proximal tubules. These FDP’s were demonstrated with anti-chain antisera, though not with antisera against 0 or E, and are presumably early FDP’s derived from the α-and β-chain. Later (at 40-120 min), FDP’s were found in liver macrophages. These FDP’s proved unreactive to anti-chain antisera, but were demonstrated by anti-D or E antisera, A similar localization of FDP’s was found in human autopsy material in cases of DIC.
Binding of Platelet Factor 4 to Cultured Human Endothelial Cells
Authors: Busch C. , Dawes J. , Pepper D.S. , Wasteson Å
Published: May 2019

Platelet factor 4(PF-4) is a product of the platelet release reaction. A well known property of PF-4 is to interact with sulphated glycosaminoglycans (CAGs), including heparin; binding to heparin leads to neutralization of the anticoagulant activity.

This study was undertaken to examine the possible binding of PF-4 to monolayers of cultured human endothelial cells(EC). The EC surface has been shown to expose GAG in particular heparan sulphate. Cultures incubated at 37°C with various amounts of 125I-PF-4 bound 125I-radioactivity in a dose-dependent manner. Maximum binding was about 500 ng per 0-7 cm2 dish (0.4x106 cells). About 50% of this was associated with the cells. In the presence of 200 ug/ml of unlabelled PF-4 or 6 ug/ml of heparin the binding was less than 5%. Chondroitin-4-sulphate (30 ug/ml) had little influence on the binding.

The observed EC-PF-4-binding in vitro may reflect a similar interaction in vivo. Such a phenomenon may influence the non-thrombogenic properties of the vascular lining. Furthermore it could affect the reliability of the PF-4 measurements as indicators of intravascular platelet release.
Acquired Storage Pool Disease in Myeloproliferative Disorders
Authors: Pareti F.I. , Mannucci P.M. , Asti D. , Guarini A. , Gugliotta L. , Tura S.
Published: May 2019

Defective aggregation and morphological abnormalities of platelets have been observed in myeloproliferative disorders, but their underlying nature is not well understood. We have examined 40 patients, 21 with thrombocythemia. 14 with polycythemia vera and 5 with myelofibrosis. All showed abnormal aggregation with ADP, adrenaline or collagen, and reduced content of platelet serotonin (5HT). ADP levels were also low, and ATP/ADP ratios correspondingly higher than normal. 5HT uptake was abnormal, and in the presence of imipramine, 14C-5HT leaked out of platelets during prolonged incubation in vitro, indicating a defect of the 5HT storage mechanism. Thromboxane B2, measured bv radio immunoassay, was produced in normal amounts when the platelets were challenged with thrombin. 5 U/ml, indicating that the prostaglandin synthesis pathway was unimpaired. The content of α-granule markers, suchas β-thromboglobulin and lysosomal enzyms, was normal in all patients, In essential thrombocythemia. polycythemia vera and in myelofibrosis, the platelet abnormality appears to be associated with an acquired defect of the storage mechanism for nucleotides and serotonin, affecting only some of the platelets. Whether this is due to the production of platelets by an abnormal stem cell line, or to exposure of platelets to inducers of the release reaction in vivo remains to be determined.
Analysis of Platelet Membrane Glycoproteins in Myeloproliferative Disoroers
Authors: Eujimura K. , Taketomi Y. , Kuramoto A.
Published: May 2019

The platelet membrane glycoproteins (GP) of myeloproliperetion disorders(12 cases) were analyzed by SDS PAGE. In GP I peak there was no difference between normal and this disorders in the 125I incorporation rate but the percentage of GP I to all PAS stained peaks was reduced in chronic myelogenous leukemia (CML) but increased in primary thrombo-cythemia(PT) polycythemia vera(PV) and myelofibrosis (MMM). In GP m peak of CML, the increased percent of PAS staining and the decreased 125I incorporation rate were proved compared to the control. In other myeloproliferative disorders, it was shown that the percent of PAS staining was normal but 125I incorporation rate was decreased against GP III. To analyse the dissociation between PAS stain and 125I incorporation rate, the two dimensional electrophoresis was conduced with non reduced-reduced system. The autradiographic pattern of this electrophoresis disclosed seven 125I incorporated spots (GP Ia, Ib, Ic, IIa, IIb, III and IV) in normal platelets. GP Ib was exposed as an unclear spot in CML (4/6). GP IV appeared as a most dense spot in control but were divided in two spotsimain large and small satellite fragment in 4 of 6 CML and in one of PV and MMM. This findingsuggested a heterogeneity of membrane glycoprotein in these disorders.
Clinical Studies of the Use of a Fluorogenic Substrate Assay Method for the Determination of Plasminogen
Authors: Triplett Douglas A. , Harms Cathy , Hermelin Lori , Huseby Rolf M. , Mitchell Gary A. , Pochron Sharon P.
Published: May 2019

Utilization of a recently reported method for the determination of plasminogen by fluorometrit technique (Ref. 3, Pochron, et. al., Thrombosis Research, Vol. 13, pg. 733, 1978) was compared with acceptable methodologies (e.g. the caseinolvtic and radial-immunodiffusion assays). Excellent results were seen with the normal range of 2.4–3.8 CTA U/ml of plasminogen by the synthetic substrate assay as compared to 8.7-14.3 mg/dl by the radial immunodiffusion assay. Normal ranges for the caseinolvtic assay were seen to be 2.2-4.0 CTA U/ml. Patients’ studies were performed on a general medical population but centered primarily on “pre” and “post” operative coronarv-pulmonary bypass patients. In all cases studied, a drop in Plasminogen level was noted in this group of patients following surgery (20–50%). Other clinical data will be presented indicating that the determination of plasminogen by the fluorometric assay procedure is sensitive and accurate for clinical situations.
Cohparison of Variability of Different Methods of Measuring Fibrinolytic Activity
Authors: Chakrabarti R. , Thompson S.G. , Meade T.W.
Published: May 2019

The Northwick Park Heart Study has drawn attention to the variability, especially within-person, of the dilute blood clot lysis time (DBCLT). However, this method has considerable practical advantages in large-scale surveys. The variability of OBCLT has therefore been compared with that of the euglobulin. lysis time (ELT) and the euglobulin fibrin plate area (EFPA). Each measurement has been made 10 times over a 9 month period in each of 12 healthy people; fibrinogen levels have also been measured. As expected, highly significant correlations (r) have been obtained between the three methods: DBCLT and ELT, + 0.63 DBCLT and EFPA, - 0.47; ELT and EFPA, - 0.54 (P<0.001 in each case). The between-pernon variance, distinguishing one person from another, accounts for 60% of the total vnriance in DBCLT, 35% in ELT and 10% in SFPA. For fibrinogen, the value is nearly 90%. The rest of the variance in each case is accounted for by hiological within-person variation and Laboratory error. Thus, while DBCLT is a variable method of determining an individual’s characteristic level of fibrinolytic activity, it is probably lees so than other commonly used methods. The results of this experiment suggest that a previous estimate of the within-person variability of DBCLT (Meade and North, 1977, British Medical Bulletin, 33, 283) may have been too high.
Kaolin-Activated Euglobulin Lysis Time (KELT)
Authors: Greig H.B.W.
Published: May 2019

The most commonly used test for clinical assessment of fibrinolytic activity is the Euglobulin Lysis Time (ELT). However the normal range is very wide, the long times are inconvenient and detection of inhibition is impossible. An attempt has been made to utilise the acceleration of the ELT when kaolin is present, to devise a test with shorter times, a narrower normal range, and better precision. The Euglobulin lysis time was carried out by a modification of the method of NILSSON and OLOW, after precipitation of the euglobulin in the absence of kaolin (ELT) and in the presence of 1 mg. kaolin/ml,, plasma (KELT). In 14 control subjects the mean, SD, and range for the ELT were 168.6’, 54.6’, 84–290’; the corresponding values for the KELT were 60.3’, 8.3’ and 46-74’. However, it was found that there was no correlation between the ELT value and the corresponding KELT (‘r’ = -0.021); on the contrary, the longer the ELT. the greater the shortening produced by kaolin and there is a direct correlation between the ELT and the shortening of the lysis time by kaolin; ‘r’ - 0.988. It is concluded that the KELT has no value as a clinical measure of fibrinolytic activity;further, the results suggest that kaolin may remove an inhibitor(s) of plasminogen activation as well as initiating Factor XII - mediated plasminogen activation.
Functional Relationship of Polymerization Sites of Vertebrate Fibrinogens
Authors: Ciernlewski C.S. , Krajewski T. , Janiak E.
Published: May 2019

Various studies on the interaction of immobilized mammalian fibrinogen and fibrin monomers with some fibrinogen derivatives demonstrated the presence of two sets of polymerisation sites In the mammalian fibrinogen molecule. We obtained the same results while investigating the fibrinogen molecules of other classes of vertebrates /Pisces, Amphibia, Aves/. Despite significant differences among their subunit structures, all of them contain polymerization sites homologous to mammalian counterparts. Moreover, due to great functional similarity, fibrinogen or fibrin monomers of the analyzed species of Pisces, Amphibia, Ayes and Mammalia interacted in a specific way with Immobilized pig fibrin monomers or fibrinogen, respectively. Using these pig affinity adsorbents, fibrinogen and fibrin monomers of different vertebrates were isolated directly from plasma and analyzed by SDS Polyacrylamide gel electrophoresis. Polypeptide compositions of eluted proteins were identical to those obtained for corresponding fibrinogen preparations isolated by cold-ethanol fractionation method. It appears to indicate that the nature of polymerization sites in vertebrate fibrinogens is alike.
The Determination of Factor X Using an Automated Amidolytic Technique
Authors: van Wijk E.M. , Kahlé L.H. , ten Cate J.W.
Published: May 2019

An automated method for the determination of factor X has been developed using a procoagulant fraction from Russell’s Viper Venom (RW), which activates FX within 4 minutes at pH 8.6, resulting in a Xa activity that is assayed using the chromogenic substrate S 2222. The amidolytic activity generated by sufficient RW is proportional to the FX level of the plasma and is not influenced by antithrombin III within the time studied. By using polybrene, the influence of heparin (up to 10 U/ml) can be eliminated. The method has been adapted for performance automatically on an Automated Kinetic Enzyme and Substrate analyser (Vitatron), Comparison of FX activities of 30 normals and 41 patients with bleeding disorders revealed a close correlation of this method with the one stage clotting assay using thromboplastin (r=0.93). Values obtained in a group of 83 patients on oral anticoagulant therapy showed good correlation (r=0.87) but tended to be about 8% higher compared to the one stage method. No significant differences were found when RW was used in the clotting assay (r-0.91). The values obtained by the amidolytic assay correlated well with thrombotest (r=0.87).
Factor X Activation by Washed Human Platelets
Authors: van Wijk E.M. , Kahlé L.H. , ten Cate J.W.
Published: May 2019

In a system of washed human platelets, Ca2+ and purified human factors X an. II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX along for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+ factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.
Factor X Vorarlberg, a New Variant of Hereditary Factor X DeficiencyX
Authors: Lechner K. , Mähr G. , Margariteller P. , Deutsch E.
Published: May 2019

The proposita, a 56 years old woman, has a mild bleeding tendency. Prothrombin time was 112 sec (control 17.0 sec), stypven time 13.7 sec (control 11.5 sec) and APTT 54 sec (control 40 sec). Factor I, II (funct.and imm.), V, VII, VIII, IX, XI, XII, AT III were normal. - Factor X activity was < 1% by extrinsic system assay (tissue thromboplastin), 12% by RVV assay and 32% by intrinsic system (APTT) assay. Similar results were obtained when factor X activity was determined using the synthetic substrate S 2222, Immunoassay (antibody neutralisation, heterologous antibody to factor X) demonstrated a decreased level (20%) of immunoreactive factor X. The patient plasma had no inhibitory activity. Factor X activity could be completely absorbed on barium sulfate and eluted with 5% sodium citrate. It was eluted at the same position from a Sephadex G-200 column as normal factor X, but nad a slower electrophoretic mobility when subjected to disc electrophoresis. Family studies (22 members studied) suggest an autosomal recessive inheritance, A second unrelated family with the same defect was subsequently detected in the same valley of Vorarlberg (Austria). - It is concluded that this hereditary coagulation disorder is Je to a grossly abnormal factor X-molecule.
The Heterogeneity of Congenital factor X deficiency. A study of two unrelated patients
Authors: Porter N.R. , Malia R.G. , Cooper P.C. , Preston F.E.
Published: May 2019

Two unrelated patients with congenital factor X deficiency are described. R.N. a four year old Pakistani child of consanguinous marriage presented with gastrointestinal haemorrhage. Routine tests of coagulation revealed a grossly prolonged prothrombin time and KCCT. All factor assays, other than factor X, were normal. Factor X, measured by three unrelated methods, was < 1%. Factor X related antigen (FXRAg) measured by antibody neutralisation was undetectable. In other, less affected members of the family FXC/FXRAg = 1 An infusion of factor X concentrate raised the factor X to 140%. The rate of decline was biphasic with an initial rapid component, T½ = 4 hours, and a slower second component T½ = 33 hours. The second patient, P.P. a Caucasian female presented with persistent post-dental extraction haemorrhage. Routine tests of coagulation revealed a prolonged prothrombin time and KCCT. All factor assays, other than factor X, were normal. Factor X, measured by two unrelated methods was 20 - 32% (normal 58- 134%) FXRAg was 71% (normal 60 - 120%). FXC/FXRAg = 0.35. Similar ratios were obtained on three other affected members of the family. The ratio represents an Important difference between the two families. Studies using the modified thrombotest strongly suggest an abnormal molecular complex in P.P. The results provide further evidence of the heterogenity of factor X deficiency states.