bluuebunny/crossref_metadata_2025 huggingface dataset:
A Chromogenic Factor X Assay with New Standardized Reagents
Authors: Friberger P. , Lenne C.
Published: May 2019

A recently published method for Factor X (FX) assay (1) utilizing Russel’s Viper Venom (RW) and a chromogenic substrate has been further investigated by testing a large number of parameters, This method has been considered as a suitable method for monitoring coumarol treatment (Bergström et al).

The conditions for the activation of FX by purified preparations of the RVV have been studied as well as the conditions for FXa determination with a new chromogenic substrate Bz-Ile-Glu(γ-piperidyl)-Gly-Arg-pNA (S-2337). Both purified factors and normal plasma have been used. The effect of plasma inhibitors as well as the selectivity of the method bas been studied.

The reproducibility and stability of the different reagents and standards have been studied and found to be good.

The amount of FXa activity obtained from normal human plasma has been titrated with FXa inhibitors of known purity.

1) Aurell L. et al, Thromb. Res., 11, 595 (1977)

2) Bergström et al, Thromb. Res., 12, 531 (1978)
Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay
Authors: Walz Daniel A. , Brown Thomas R.
Published: May 2019

Human prothrombin activation is unique in that, in addition to the release of fragment 1-2 (F1·2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed”from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prothrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400of plasma. Serum, obtained from whole blood clotting, conr tained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents on 5–10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure Is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).
Bleeding Complications in Treatment with ASA and Anticoagulants
Authors: Dale J. , Myhre E.
Published: May 2019

Patients with prosthetic heart valves have a reduced platelet adhesiveness and a high incidence of arterial thromboembolic episodes. Therefore 148 patients with aortic ball valve prostheses were chosen for an antithrombotic study. They received either one gm. of ASA or placebo in combination with anticoagulants. In two years, 15 bleeding episodes developed in those taking ASA, seven during the firstmonth. Two had intracranial hemorrhage, one died, while the others recovered completely. Six episodes occurred in the placebo group, three intracranial complications caused two deaths. The higher incidence of bleeding in the combined group was entirely due to gastrointestinal hemorrhage. In both groups, the occurrence and severity of bleeding correlated to the intensity of anticoagulant therapy. Low platelet adhesiveness also predisposed to bleeding.
The Role of Fibrinolysis in Pathogenesis of Chronic Glomerulonephritis
Authors: Andreenko G.V. , Podorolskaya L.V. , Polentseva L.R.
Published: May 2019

The levels of fibrinolytic activators and antithrombins were examined in the blood and urine samples of 71 patients with various forms of chronic glomerulonephritis (CGN) before and 20 min after venous occlusion. The release of heparin and plasminogen activators into the blood stream and the increase of antithrombin III and non-enzymatic fibrinolysis were observed only in the patients with a latent form of CGN. The fibrinolytic activity of urine was increased with an increase in urokinase and other activators and a decrease of the urokinase inhibitors. In patients having more severe nephrotic forms of CGN with thrombotic complications the venous occlusion was followed by a decrease in concentrations of heparin and plasminogen activator in the blood and urine and by a decrease of the non-enzymatic fibrinolytic activity of the blood. A retrospective analysis of 41 patients with a nephrotic form of CGN showed that a favourable outcome with remission was observed only in the patients having an adequate response to the venous stasis (i.e. release of activators and hecarin).
Dysfibrinogenemia and Hypercoagulability in Patients with Membranous Glomerulopathy
Authors: Winter C. S. , Wagoner R. D. , Bowie E. J. W. , Owen C. A.
Published: May 2019

Renal vein thrombosis (RVT) is a common complication in patients with nephrotic syndrome and membranous glomerulopathy (MGN). We demonstrated it angiographically in 12 of 24 consecutive patients. Thorough hemostatic surveys were done on 14, 6 with RVT. In 11 patients, 5 with RVT, platelet counts were significantly above normal, 402,000 to 700,000/mm3 blood. In all but 4 cases, plasma fibrinogen was at least twice normal (mean 588 mg/dl). Factor VIII: C levels ranged from 152 to 337 U/dl (mean 236). Antithrombin III was depressed in only one patient, lie did not have RVT. While prothrombin times, partial thromboplastin times, and activated partial thromboplastin times were normal in all patients (except when anticoagulants were being given), the thrombin time was significantly prolonged in 12 of the 14 patients (6 with RVT). In these 12 the Reptilase times were also long. The prolonged thrombin times cannot be attributed to inhibition by fibrinolytic degradation products since the FDP were elevated in only 5 cases (2 with RVT). It seems that an abnormality of the fibrinogen molecule (prolonged thrombin and Reptilase times) and “hypercoagulability” characterize the nephrotic syndrome associated with membranous glomerulopathy and possibly other renal lesions. We could not, however, distinguish between patients with and without renal vein thrombosis.
Platelet Stimulation by Tetranitromethane-Modifled α-Thrombin
Authors: Larsen N.E. , Simons E.R. , White G.C. , Lundblad R.L.
Published: May 2019

Stimulation of washed platelets by α-thrombin is accompanied by a dose-dependent depolarization of the platelet membrane, Tosyllysylchloromethylketone-treated thrombin (TLCK-thrombin), modified at the active site, is enzymically inactive and cannot induce such depolarization but can partially inhibit the thrombin-induced membrane potential change, the extent of such inhibition depending on the TLCK-thrombin: α-thromb in ratio. In the present study we have performed comparable investigations using thrombin modified at the macromo lecular binding site with tetranitromethane (TNM-thrombin) which is almost fully active against small substrates like tosylarginylmethylester, but inactive against macromolecular substrates like fibrinogen. TNM-thrombin stimulated platelet membrane depolarization only at a dose 10X that of α-thrombin. Preincubation with TNM-thrombin reduced the subsequent response to α-thrombin, the total response being somewhat lower than that achieved for the control alone at an identical concentration. These results are consistent with the hypothesis that platelet membrane depolarization induced by thrombin is dependent on its proteolytic active site. Modification of the macromolecular binding site impairs thrombin binding to the platelet surface, leading to diminished membrane potential change, and thus supporting the correlation between binding of thrombin to the platelet surface and platelet membrane depolarization, Supported in part by NIH grants HL-15335 and DE-02668 as well as AHA Grant-in-Aid 78-1215.
Thrombin-Induced Surface Changes of Human Platelets in Plasma: Their Relation to Serotonin Release
Authors: Subbarao K. , Kakkar V.V.
Published: May 2019

Membrane proteins of both control and thrombin-treated platelets were labeled by NaB3H4, reduction of Schiff bases formed between pyridoxal 5′-phosphate and protein amino groups. Examination of the labeled polypeptides by SDS-polyacrylamide gel electrophoresis and fluorography disclosed a different labeling pattern for thrombin-treated platelets. The distributions of Coomassie blue-stained protein from treated and untreated cells were, by contrast, almost identical. Fluorographs of control platelets showed a single intensely labeled protein band (mol wt 90,000) whereas with cells exposed to thrombin (30-60 milliunits) about 10 protein bands with mol wts ranging from 43,000 to 200,000 were typically present. Among these were: thrombin-sensitive protein (mol wt 188,000), glycoprotein I (mol wt 150,000) and actin (mol wt 43,000). When serotonin release was prevented, either by reversing platelet aggregation with low amounts of ADP (0.1-0.3 μM) or by preincubating with 3',5'-ADP (20 μM), an inhibitor of both ADP- and thrombin-induced platelet function, the labeling patterns on fluorographs were similar to the control. These results indicate that blood platelets can undergo reversible aggregation without major changes in their surface topography, whereas thrombin-induced serotonin release appears related to structural alterations in platelet membrane proteins.
Plasma Thromboxane B2(TxB2) and Fibrinopeptide A (FpA) in Patients with Thrombosis and During Contact of Blood with Artificial Surfaces
Authors: Davies G.C. , Sobel M. , Salzman E.W.
Published: May 2019

To study thrombin activity and platelet stimulation during interaction of blood with artificial surfaces, we measured circulating FpA (normal up to 2.3 ng/ml) and TxB2 (normal 0.08-0.32 ng/ml) by radioimmunoassay. 31 of 32 patients referred for phlebography for suspected DVT had elevated FpA (mean 49 ng/ml) but normal TxB2. 23 patients had DVT and the others had ruptured Baker’s cyst, muscle tears, hematomas, and inflammatory conditions. FpA rose further after phlebography (mean rise 34.6 ng/ml); similar changes followed arteriography and cardiac catheterization. Heparinization failed to return elevated FpA to normal. None of the procedures affected TxB2 levels.

General surgical operations raised FpA but not TxB2. However intra-aortic balloon pumping elevated TxB2 (max. rise 0.44 ng/ml). In 24 of 29 patients undergoing cardiopulmonary bypass using a bubble oxygenator, TxB2 rose over 0.05 ng/ml within the first 120 min. of bypass (mean rise 0.49 ng/ml, max. rise 2.0 ng/ml). The levels detected imply sufficient in-vivo thromboxane A2 production to cause platelet aggregation and myocardial ischemia from coronary artery constriction. FpA levels were elevated even before bypass, probably because of intravascular cannulae, and did not return to normal despite “adequate” heparinization (judged by Activated Clotting Time), implying continued thrombin activity during bypass.
Potentiation of the Anti-Aggregating Activities of Prostacyclin by Verapamil
Authors: Ikeda Y. , Toyama K. , Hasegawa M. , Yamamoto M. , Watanabe K. , Ando Y.
Published: May 2019

Prostacyclin (PGI2), which is a potent inhibitor of platelet aggregation formed in vascular wall, is now considered to play a crucial role in the pathogenesis of thrombosis. Evidence was presented in this investigation that a coronary vasodilator, Verapamil (VP), markedly potentiates the anti-aggregating activities of PCI2. VP caused a dose dependent inhibition of serotonin release induced either by thrombin or by A23187 in the absence of extracellular calcium. Addition of 1 mM CaCl2, with A23187 or thrombin partially overcame this inhibition, while 1 mM CaCl2 alone caused no significant release in the absence of A23187 or thrombin. Minimal concentration of PGI2 (supplied from Hoechst Research Instituted West Germany) to cause 50% inhibition of collagen-induced platelet aggregation was 1 nM, while that of VP was 100 μM. Simultaneous addition of 0.125 nM PGI2 and 30 μM VP resulted in complete inhibition of aggregation. Similar synergistic effects between PGI2 and VP were observed in platelet aggregation induced by ADP, arachidonic acid and A23187. 8-(N-N-diethylamino)-octyl 3,4,5-trimethoxybenzoate (TMB-8), a compound recently show to act as an intracellular calcium antagonist (I. Charo, Bioch. Biophy. Res. Comm.), also potentiates the inhibitory effect of PCI2 on platelet aggregation.

Our results suggest that an intracellular calcium antagonist may possibly become an unique anti-platelet agent because of its ability to inhibit release reaction and to potentiate the anti-aggregating activity of PGI2.
Carbenicillin and Penicillin G Impair Platelet Function by Inhibiting the Binding of Agonists to the Platelet Surface
Authors: Shattil S.J. , Bennett J. , McDonough M. , Turnbull J. , Vilaire G.
Published: May 2019

Carbenicillin (carb) and penicillin G (pen) inhibit human platelet function in vivo and can cause a bleeding diathesis. Since the mechanism by which these drugs inhibit platelets is unknown, we investigated whether they might interfere with the binding of epinephrine, ADP, or ristocetin (von Willebrand factor) to the platelet surface in vitro. Pen (10 mM) or carb (20 mM) produced a 6-fold decrease in the affinity of receptors on intact platelets for both epinephrine and for the α-adrenergic antagonist, 3U-dihydroer-gocryptine (p < 0.001). Neither antibiotic changed the maximum number of α-adrenergic binding sites per platelet. Pen and carb at these concentrations completely inhibited 14C-serotonin release induced by 1 μM epinephrine and inhibited primary platelet aggregation by 1 μM. Similarly, these antibiotics inhibited aggregation and serotonin release induced by 1 μM ADP and inhibited the covalent binding of an ADP analogue (5'FSO2BZAdo) to its specific binding protein in platelet membranes. Moreover, pen and carb inhibited competitively the agglutination of platelets by ristocetin. Thus, carbenicillin and penicillin G decrease the affinity of a number of distinct platelet receptors for their specific agonists, and this correlates with inhibition of platelet function induced by these agonists. Interaction of these antibiotics with the platelet surface membrane may account for their hemorrhagic as well as their anti-thrombotic activity.
An Abnormal Fibrinogen with Delayed Fibrinopeptide a Release
Authors: Lane D.A. , VanRoss M. , Kakkar V.V. , Dhir K. , Holt L.P.J. , MacIver J.E.
Published: May 2019

An investigation has been carried out of a coagulation defect occuring in three generations of a family as an autosomal dominant trait. Coagulation tests revealed a slightly elevated fibrinogen concentration, a normal FDP level yet prolonged thrombin clotting time, suggesting a congenital dysfibrinogenemia. Further plasma clotting studies demon strated no pH dependence of the defect, little correction by calcium ions, but a total correction when normal and patient plasma were mixed in equal amounts. A dysfibrinogenemla was confirmed when fibrinogen purified from plasma of one of the patients exhibited delayed clotting with thrombin. A small delay in the rate of polymerisation of monomers was evident when purified patient monomers were compared with those of normals. Electro-phoretic, immunoelectrophoretic and immunodiffusion experiments were able to detect neither structural nor immunological abnormalities associated with the intact molecule nor with the A α, Dβ, and γ chains of fibrinogen. Factor XIIIa induced cross linking of fibrin appeared normal as judged by SDS Polyacrylamide gel electrophoresis. However, using a specific radioimmunoassay to fibrinopeptide A the major defect has been Localised to a delay in the rate of release of this peptide by thrombin when the patient fibrinogen is converted to fibrin. The abnormality has been tentatively designated fibrinogen Manchester.
Defective Thrombin Binding by Abnormal Fibrin Associated with Recurrent Thrombosis
Authors: Liu C.Y. , Nossel H.L. , Kaplan K.L.
Published: May 2019

Studies of clotting activity and radioactivity (with 125I-thrombin) indicated that binding of human thrombin to fibrin depends upon the initial concentrations of the reactants. Scatchard analysis suggests two classes of binding site: a high affinity site with a Ka 5.8±0.9 x 105M-1 and a maximum molar binding ratio of thrombin to fibrin 0.4±0.2, and a low affinity site with a Ka 6.8±0.6 x 104M-1 and a maximum molar binding ratio of 1.6±0.5. The active site of thrombin is not required for binding since neutralization with phenylmethyl sulfonyl fluoride does not affect the binding. Thrombin also binds to fibrin formed in whole blood and can be recovered with full clotting activity when the clot is dissolved by plasmin, suggesting that binding of thrombin to fibrin is a potential mechanism for limiting thrombosis. Fibrinogen New York I, in a patient with recurrent venous and arterial thrombosis, was characterized by abnormalities in fibrinopeptide release by thrombin and fibrin polymerization. The patient’s plasma contained equal amounts of normal and abnormal fibrinogen. Thrombin was bound normally by fibrin derived from the normal fibrinogen but was not bound at all by fibrin from the abnormal fibrinogen. Fibrinogen New York II, in an asymptomatic patient, was characterized by delayed fibrinopeptide release and fibrin polymerization. Thrombin was bound normally by this fibrin. Thus defective binding of thrombin by fibrin is suggested as a new mechanism predisposing to recurrent thrombosis.
Modulator Binding Protein in Platelets
Authors: Muszbek L. , Hàrsfalvi J.
Published: May 2019

In a previous paper (Muszbek et al. FEBS Letters 1977, 80, 308) the existence of a troponin C-like protein has been revealed in platelets. This protein was isolated, characterized and identified as the multifunctional Cadependent regulator protein (modulator). Here we show that there is a platelet protein which in 6·3 M urea if Ca2+ present can form a complex with modulator protein and with skeletal muscle troponin C, too. It withstands aceton treatment and can be extracted from platelet aceton powder by 1 M KCl. Further purification could be achieved by affinity chromatography on an Affi-Gel 10-troponin C column. Modulator binding protein also copurified with platelet actomyosin though there was no detectable amount of modulator protein in this preparation. Since modulator protein appears to be responsible for the Ca2+ regulation of purified platelet myosin light chain kinase (Dabrowska and Harsthorne BBRC 1978, 85, 1352) the low Ca2+ sensitivity of platelet actomyosin may be due to the virtual absence of modulator and/or to the presence of modula tor binding prote in in thrombosthenin.
Heparin Induced Release of Low Affinity Platelet Factor Four
Authors: Rao A.K. , James P. , Niewiarowski S.
Published: May 2019

Heparin has been reported to effect platelet function. We studied the effect of commercial beef lung heparin on the release of a platelet specific protein, the low affinity platelet factor 4(LA-PF4) in vitro and vivo. LA-PF4 shares Common antigenic determinants with B thromboglobulin, LA-PF4 was measured in platelet poor plasma (PPP) by radioimmunoassay. The mean levels of LA-PF4 in PPP from citrated blood and citrated blood with added heparin (final concentration 1.5 unit/ml) were 78.3±18.3 and 242.5±117.1 ng/ml respectively. (Mean ± SD, p <0.01). Addition of heparin to blood collected in a mixture of E EDTA, prostaglandin El, and theophylline did not result in an increase in LA-PF4 in PPP. A single intravenous bolus of heparin was administered to two normal individuals and the levels of LA-PF4 measured before and at 15,30 and 180 minutes later. An increase in the levels of LA-PF4 in the PPP from a baseline of 66.7 and 30.2 ng/ml/106 platelets to 166.7 and 167.4 ng/ml/106 platelets respectively were noted in 30 minute sample. There was no increase in LA-PF4 at 15 minutes. At 180 minutes levels were at baseline. It is concluded that heparin stimulates release of LA-PF4 from platelets both in vitro and in vivo. LA-PF4 has antiheparin action and is being investigated as a marker for thrombosis. Its release by heparin may, therefore, have an important implication.
Roles of Cyclic Nucleotides in the Inhibition of Platelet Function by Physiolocic and Pharmacological Agents
Authors: Haslam R.J.
Published: May 2019

Cyclic AMP mediates the inhibitions of platelet aggregation caused by PCI2, PGE1 and PGD2. Thus, these compounds activate platelet adenylate cyclase and Increase platelet cyclic AMP; their inhibitory effects are blockod by inhibitor? of adenylate cyclase, are potentiated by inhibitors of cyclic AKP phosphodiesterase and are mimicked hy N6 ,2'-0-dibutyryl cyclic AMP. Inhibition of adenylate cyclase does not potentiate platelet aggregation in the absence of inhibitory prostaglandins, indicating that platelet cyclic AMP is too low to affect aggregation under these conditions. To determine whether platelets in the circulation are exposed to agents that increase platelet cyclic AMP, washed rabbi platelets labelled with [3H] adenine were incubated with rabbit arterial blood under various conditions; any increases in cyclic [3H]AMP were measured. These experiments showed that freshly taken rabbit arterial blood does not normally contain any factors that can increase platelet cyclic AMP sufficiently to affect platelet function; specifically, circulating PGI2 was less than 0.1 pmol/ml of blood. It follows that increases in cyclic AMP in circulating rabbit platelets must occur only locally or under special conditions. The role of the moderate increases in platelet cyclic CMP caused by aggregating agents remains uncertain, but the inhibition of aggregation by compounds such as sodium nitroprusside that increase cyclic CMP up to 100-fold suggests that cyclic CMP may, like cyclic AMP, be an inhibitory mediator.
A Class of Drugs that Inhibits Platelet Release and Aggregation by Apparent Interference with Anion Transport
Authors: Shulman N.R. , Pollard H.B. , Tack-Goldman K.
Published: May 2019

The platelet release reaction is analogous to the process of exocytosis by which many other secretory cells release hormones or mediators from intracellular granules. Anion transport blocking (ATB) drugs Inhibit release of epinephrine from isolated chromaffin granules (CG) by blocking chloride uptake and preventing osmotic lysis. Studies on platelets analagous to those done on CG showed that increasing osmotic strength in the range 600-1000 m0sm progressively suppressed serotonin release to completion and that ATB drugs (viz, probenecid, SITS, pyridoxal phosphate and suramin) at mM concentrations completely inhibited release and aggregation of human platelets stimulated by thrombin, ADP, A23187, epinephrine or collagen. Sulfinpyrazone has the appropriate structure for anionic blocking, and may suppress platelet function as effectively by this mechanism as by cycloxy-genase inhibition. The ATB drugs acted apparently to prevent movement of OH- from the more alkaline medium into the relatively acidic granule, for platelet release was not inhibited by replacing anions with isethionate or sucrose, but was markedly dependent on OH- in the pH range 6 to 7.5 where inhibition by the ATB drugs was competitive with respect to OH-. Since the ATB compounds include some relatively nontoxic drugs in common use, and since their action on platelets differs markedly from that of aspirin, they should receive attention as potential alternative or auxiliary antithrombotic agents.
Structural Aspects of Calcium Binding to Fibrinogen
Authors: Marguerie G.A.
Published: May 2019

The important role of Ca++ in the clotting of fibrinogen has been known for some time. In order to elucidate the mechanism by which this cation influences the structure and the function of this protein the Ca++ -fibrinogen interaction has been examined. Equilibrium dialysis experiments at pH 7.4 have shown that fibrinogen-possesses three Ca++ binding sites of high affinity with an apparent association constant Kd = 2 × 10-6 M. The specificity for Ca++ was established and a partial identification of the binding sites demonstrated that, Ca++ is tightly bound to the protein through a chelate system. Binding studies at different pH also demonstrated that these sites do not behave as homogeneous population and at least two distinct classes of sites could be demonstrated. Whereas physicochemical analysis established that the binding of Ca++ to fibrinogen does not entail a gross overall conformational change, protection against denaturation could be demonstrated indicating that Ca++ is part of the structure of the fibrinogen molecule and might stabilize a more compact molecular conformation which is structurally and functionally different. This was corroborated by studies of the influence of this cation on two basic reactions of fibrinogen, namely proteolysis by thrombin and plasmin. It was found that: 1) resistance to proteolysis by plamin is observed when Ca++ is bound to fibrinogen. The rate of the cleavage of the earliest plasm in-susceptible bound is reduced, 2) Ca++ catalyzes the proteolytic reaction between thrombin and fibrinogen. Based upon these observations a proposal is tentatively made to explain the role of Ca++ on the structure:function relationship of fibrinogen.
Arrangemet, of Monomeric Units in Fibrin
Authors: Hermans Jan
Published: May 2019

Measurements of light scattering have given much information about formation and properties of fibrin. These studies have determined mass-length ratio of linear polymers (protofibrils) and of fibers, kinetics of polymerization and of lateral association and volume-mass ratio of thick fibers. This ratio is 5 to 1. On the one hand, this high value suggests that the fiber contains channels that allow the diffusion of enzymes such as Factor XIIIa and plasmin; on the other hand, the high value appears paradoxical for a stiff fiber made up of elongated units (fibrin monomers) arranged in parallel. Such a high fiber volume is a property of only a small set out of many high-symmetry models of fibrin, which may be constructed from overlapping three-domain monoirers which are arranged into strands, are aligned nearly parallel to the fiber axis ant) make adequate longitudinal and lateral contacts. These models contain helical protofibrils related to each other by rotation axes parallel to the fiber axis. The protofibrils may contain 2, 3 or 4 monomers per helical turn and there are four possible symmetries. A large specifi volume is achieved if the ends of each monomer are slightly displaced from the protofibril axis, either by a shift or by a that of the monomer. The fiber containing tilted monomere is more highly interconnected; the two ends of a tilted monomer form lateral contacts with different adjacent protofibrils, whereas the two ends of a non-tilted monu mer contact the same adjacent protofibril(s).
In Vitro Synthesis of Bovine Prothrombin in a Purified System
Authors: de Metz M. , Vermeer C. , Soute B.A.M. , Hemker H.C.
Published: May 2019

Coumarin treatment of the cow results in the appearance of a prothrombin precursor (de-carboxyprothrombin) in the blood. We have isolated from bovine liver the enzyme (prothrombin synthase) that is able to convert purified decarboxyprothrombin into prothrombin. Prothrombin synthase is localized in the microsomal fraction and extracted there from with a buffer containing 2% Triton X-100. Further purification of the enzyme was accomplished by column chromatography on Sepharose 4B, DEAE Sephacell and Sephadex G 100. The molecular weight of prothrombin synthase was found to be about 60,000 D. The enzyme activity is defined as the ability to increase the prothrombin concentration at standard conditions and it is measured as a decrease of the clotting time in the one-stage coagulation assay. In the presence of NaH14CO3, radioactive label is incorporated into decarboxyprothrombin, parallel to the generation of prothrombin.

After extraction with a suitable organic solvent prothrombin synthase was separated from endogenously bound vitamin K. After extraction the water phase contained the enzyme, the activity of which was completely restored by supplementing the reaction mixtures with vitamin KH2. The organic phase contained the vitamin K as was deduced from U.V.spectra. Prothrombin synthase is dependent on Mn2+ and molecular oxygen and is inhibited by warfarin, Cl-K and Cu2+.
Evidence that Thrombin Releases Fibrinopeptide B From non-Polymerizing Fibrin
Authors: Brosstad F. , Kierulf P. , Gravem K. , Godal H.C.
Published: May 2019

When thrombin acts upon fibrinogen (Fg), substantial amounts of fibrinopeptide A (FPA) are released prior to significant release of fibrinopeptide B(FPB). It has been claimed that polymerization of des-AA fibrin is a prerequisite for the release of FPB from des-AA fibrin, by exposing structures (conformational changes) required for the FPB part of the B beta chain to be recognized by thrombin (Blombäck, Hogg and Blombäck, 1976, 1978). To test this theory, either Fg or des-AA fibrin(made by exposure of Fg to insol. reptilase in urea) were exposed to thrombin (l or 100 NTH units) in a non-polymerizing system (2.5 M urea, pH 7.4,I=0.15, temp.+20°C), measuring FPA release(FPA-RIA +N-terminal analysis) and FPB release (N-terminal analysis). At 1 NIH unit of thrombin, complete release of FPA from Fg in urea occurred, whilst no FPB release from Fg or des-AA in urea were noted, irrespective of the incubation time,(Potency of thrombin during incubation time was unchanged). Incubation with 100 NIH units of thrombin caused complete release of both FPA and FPB from 30 mg of Fg or des-AA fibrin in urea within 5 min, and chromatography of the resulting des-AABB fibrin, (80 × 2 cm, Sepharose 4B, 2.5 M urea) demonstrated a homogenous elution profile, indicating absent polymerization. It seems likely that urea in some way interferes with the affinity of thrombin for FPB, which might be compensated for by the use of high thrombin concentrations. It is concluded that polymerization is not a prerequisite for the release of FPB by thrombin.
A New Radioimmunoassay-Technique for Fibrinopeptide-a in Human Plasma
Authors: Harenberg J. , Zimmermann H. , Haas F. , Schmidt-Gayk H.
Published: May 2019

Fibrinopeptide-A (FpA) is thought to be the most sensitive parameter to indicate hypercoagulability with increased fibrin formation in man. Previously described radioimmunoassays for FpA are very time consuming. We present therefore a modification of the assay, which is less time consuming, sensitive, reproducable and reliable. The following modifications were made on the original method: 1. plasma dialysis was omitted 2. performance on microtiter-plates 3. double antibody method.

The sensitivity was improved to 0.16 ng FpA/ml plasma. The mean concentration of FpA in dialysed and undialysed plasma did not differ significantly (p<0,001). Effective separation of fibrinogen was achieved by aethanol extraction alone. The mean concentration of FpA correlate, highly, when the tracer was separated by charcoal or second antibody (r = 0.96). The recovery was improved to 85%.

In healthy persons 0.16-2.5 ng FpA/ml plasma were measured. In patients with venous thrombosis or myocardial infarction in the history FpA was elevated significantly to 2.0 ng/ml-19.6 ng/ml, indicating that the FpA is a usefull tool in diagnosis of hypercoagulability.
The Effect of Sulfinpyrazone, Aspirin and their Metabolites on Prostacyclin Production by Rabbit Aortic Rings
Authors: Westwick J. , Webb H. , Lewis G. P.
Published: May 2019

Since sulfinpyrazone (SU) and aspirin (ASA) axe used as anti-thrombotic compounds, it is important to know the effects of these agents and their metabolites on prostacyclin (PGI2) generation. Rabbit aortic rings (RAE) incubated with arachidonic acid (AA) generate a product that inhibits platelet aggregation. That the ring product was PGI2 was shown by 1) a similar temperature-dependent decay curve when assayed against authentic PGI2, 2) the formation of the product was blocked by 15-OOH AA (10μpg/ml), 3) the action of the product on platelets was antagonised by antisera to 6-keto-PGF1α which antagonised authentic PGI2 but not PGD2 or PCE1. The optimal conditions for PGI2 generation by RAR were determined by varying storage and incubation media, time and temperature of incubation and substrate concentration. RAR (10-20mg) were incubated in 300μl buffer with each concentration of drug in quintuplicate for 5 min at 25°C. AA was added, 5 mm later a 200μ1 aliquot was withdrawn and its ability to inhibit ADP-induced platelet aggregation was compared with that produced by vehicle-treated RAR and to authentic PGI2. IC50q (μg/ml) of PGI2, production were: SU (210), sulfoxide H of SU (60), p-hydroxy M of SU (inactive), ASA (140), salicylic acid (inactive). Therefore the concentrations of sulfinpyrazone and aspirin necessary to inhibit PGI2 production are higher than those which produced their anti-thrombotic effect.
Pharmacological Distinction Between Sulphinpyrazone and Inhibitors of Prostaglandin Synthesis
Authors: Butler K.D. , Smith J. , White A.M.
Published: May 2019

Injection (i.v.) of 0.5 ml anti-Forssman antiserum to guinea pigs resulted in an irreversible thrombocytopenia (95%) but a transient thrombocytopenia (50%) ensued after 0.2 ml which was complement dependent. During the onset of the thrombocytopenia there was marked platelet sequestration in the vasculature of the lung which was reversed when the circulating platelet count returned to normal. This sequestration was associated with a biphasic bronchospasm which was both platelet and complement dependent. Sulphinpyrazone (100 mg/kg i.v.) administered 30 or 60 min before the antiserum inhibited the thrombocytopenia by 59% and 100% respectively, as we the as the platelet sequestration in the lung and the bronchospasm. Aspirin (100 mg/kg i.v.) and indomethacin (10 mg/kg i.v.) inhibited the bronchospasm but not the thrombocytopenia, while phenylbutazone was inactive in all cases. This model thus distinguishes between sulphinpyrazone and the other inhibitors of prostaglandin synthesis.
Studies on Nature of So-Called Antiplasmin Measured by Chromogenic Substrate S-2251 and its Automatic Determination with ABA 100
Authors: Kato M. , Fujimaki M. , Fukutake K. , Maki A. , Hibino M.
Published: May 2019

Using chromogenic substrate S-2251, authors studied on the nature of so-called antiplasmin and on the application of Coatest to automatic determination. As to several protease inhibitors, correlation performed vs single radial immunodiffusion yielded a regression of Y=19.5X-18.6 and r=0.91 for α2 PI;r=-0.15, 0,13, 0.46 0.28 for α2M, α1AT, AT-III and CIINA respectively. Purified protease inhibitors except α2PI did not exhibit immediate inhibition to human plasmin. The automation of chromogenic substrate method was studied with ABA Bichromatic Analyser Model 100 and auxiliary dispenser. 25 microliter of diluted plasma and 250 microliter of plasmin of known activity were added simultaneously to 100 microliter of substrate S-2251. Reaction rate was assayed with the use of two wavelengths, 450/415. Excellent correlation with manual protocol was observed, r=0.985 and Y=0,88X=8.7. Determination of within-day precision of automatic assay for three sample concentrations (Antiplasmin 252, 100%, 150%) gave C.V. of 1.5%, 1.71% and 4.46% respectively. Thus, authors arrived at the conclusion that 1) OfiPI is mainly determined as protesae inhibitor with the use of S-2251, 2) ABA 100 was successfully employed for automatic determination of antiplasmin witli the use of simultaneous addition of plasma and plasmin to substrate solution.
A New Automatic Instrument to Investigate the Rheological Behaviour of Red Blood Cells
Authors: Teitel P. , Mussler K.
Published: May 2019

Based on the principles already described (Blood Cells, 1977, 3 55) a completely automatic instrument has been constructed for the investigation of the flow behaviour of packed red blood cells at low shear stresses (below 1 Pa). Functioning as a simple poly-mi croviscometer the instrument features :(1) four cylindrical sample holders with filter mountings, (2) four opto-e Iectronic level measuring devices, (3) a microprocessor controlling the functions of the whole instrument (including the successive scan of the samples in discrete steps), (4) a digital display of the actual sample levelin each holder, (5) a memory unit to store the scanning results and (6) a logarithmic chip. At the end of the investigation the data stored can be retrieved either in analog or in digital form. The analog retrieval over a Potentiometrie recorder generates a “filtration curve” on which the type of flow behaviour (Newtonian or more or less non-Newtonian) can be directly visualieed. The digital data retrieved over a printer can be fed into a computer to calculate the equation of the flow curve. The instrument can provide information either on the forces of aggregation between individual erythrocytes or on their rigidity (washed RBC packed in buffered isotonic saline containing 1 % human albumin). These information might help iluminate the contribution of RBC Theological factors in the pathogenesis of vascular occlusion.
Measurement of Endotoxin Levels in Plasma Using Limulus Amoebocyte Lysate and Chromogenic Substrate, S2222
Authors: Clark S.E. , Scully M.F. , Webb P. , Kakkar V.V.
Published: May 2019

A method has been devised for the measurement of endotoxin in plasma using the chromogenic substrate, S2222, a substrate which has been shown to be particularly sensitive to the Limulus Lysate. Time curves of the rate of release of the chromogen in mixtures In which procoagulase activation was concurrent, were complex with a lag phase which was shortened by increasing endotoxin concentrations. At a final concentration of 0.5ng/ml and 370 activation was complete within 60 minutes. The enzyme was inhibited by soya bean trypsin inhibitor but not by pancreatic trypsin inhibitor or hirudin. In the method finally adopt ed the lysate (25μl) was incubated with endotoxin (E. coli 026.86 Difco) and magnesium chloride (final concentration 33mM) in a total volume of 225μl. After 12 minutes preincubation 165μl of S2222(0.4mM) was added and the increase in abdorbance at 405nm over two minutes measured using an Abbott Biochromatic Analyser 100. Linear assay curves were obtained with final concentration of 0.2 to 2.0ngs endotoxin/ml with ΔOD 405/min of 0.35 and 2.0ng endotoxin/ml of final incubation mixture. ×OD /min in control tubes were of the order of 0.02. For measurement from plasma samples, endotoxin was first extracted with chloroform. Linear calibration curves were achieved at a concentration of endotoxin of 1 to Sng /ml of whole blood with a net OD/min at the highest concentration of 0.25.
The Relation Between VIIIR:AG and VIII:C Studied with two Antisera
Authors: Muller H. P. , van Tilburg N.H. , Bertina R.M. , Voltkamp J. J.
Published: May 2019

FVIII was separated into low molecular weight FVIII (LMW FVIII) and high molecular weight FVIII (HMW FVIII) by gel chromatography in the presence of high salt concentration or by high salt elution of LMW FVIII from FVIII bound to anti HMW FVII-Sepharose. Specific antibodies were raised in rabbits against HMW FVIII and LMW FVIII. Mter rsroval of the contaminating anti HMW activities the rabbit anti LMW FVIII was still able to neutralize the FVIII coagulant activity of normal plaana and of LMW FVIII with comparable efficiency and it had no effect on the VIIIR:WF of FVIII in normal plasma or in HMW FVIII. Anti LMW FVIII does not bind to HMW FVIII and does not precipitate FVIII as tested by counter Immunoelectrophoresis. Rabbit anti HMW FVIII precipitates FVIII in normal plasma, inhibits VIIIR:WF activity, while it has no effect on the FVIII coagulant activity of LMW FVIII. The coagulant activity of FVIII in normal plasma is slightly inhibited by anti HMW FVIII presumably by non-specific effects (sterical hindrance). It is concluded that inhibitory antibodies against VIII:C raised in rabbits recognize antigenic structures only present on LMW FVIII. Antibodies against HMW FVIII raised in rabbits appears to recognize structures only present on HMW FVIII.
Thrombotic Thrombocytopenic Purpura: Mechanism for Effectiveness of Plasmapheresis
Authors: Kelton J.G. , Neame P.B. , Walker I. , Turpie A.G. , McBride J. , Han P. , Nicholson W. , Hirsh J.
Published: May 2019

Thrombotic thrombocytopenic purpura (TTP) is a rare but serious illness of unknown etiology. Treatment by plasmapheresis has been reported to be effective but the mechanism for benefit is unknown. We have investigated the effect of plasmapheresis in 2 patients with TTP by guantitating platelet associated IgG (PAIgG) levels prior to and following plasmapheresis. Both patients had very high levels of PAIgG at presentation (90 and 48 fg IgG/platelet respectively, normal 0-5). In both, the PAIgG levels progressively fellto within the normal range and the platelet count rose following plasmapheresis. One patient remained in remission with normal platelet counts and PAIgG levels. The other relapsed after plasmapheresis and the PAIgG levelrose prior to the fall in platelet count. Plasmapheresis was repeated and resulted in normalization of both the platelet count and PAIgG level. It is suggested that plasmapheres is removes antiplatelet antibody or immune complexes which may be of etiologicalimportance in this illness.
The Effects of PGI2 on Thrombin, Factor Xa, Plasmin and on their Inactivation by Antithrombin-III and Heparin
Authors: Blaskó Gy.
Published: May 2019

The data presented by the author demonstrate the effect of PGI2 on the activity and the inactivation of enzymes participating in blooa clotting /thrombin and Factor Xa/ and fibrinolysis /plasmin/. According to his resulte PCI2 has no effects on the activity of Factor Xa and plasmin and on the inaotivation of these enzymes by antithrombin-III and/or heparin. An acceleration of the inactivation of thrombin by antithrombin-III was found in the presence of PGI2, within a concentration range of 50-250 μg.ml. This heparin-like effect of PGI, is demonstrable only at a concentration of 5 magnitude higher than that of the antiaggregation effect on platelets /5-10 ng per ml/. Factors influencing the inactivation of thrombin and Xa by anti-thrombin-III and heparin /proteins, ions/ are under discussion.
Zonal Immobilization Procedure for Chromatographic Analysis of Fibrin Complexes
Authors: Shainoff John R. , Dardik Beatriz N.
Published: May 2019

Conventional gel filtration methods for detection of fibrin complexes depend on fraction collection and multiple analyses of the eluted protein to characterize the chromatogram. A new approach has been made possible through invention of a system for attaching the separated protein to the gel and staining it with either fluorescent or radioiodinated antibody. In this system, glyoxyl agarose (agarose-O-CH2-CHO) is used as the gel filtration medium to separate the proteins, and is subsequently activated to Immobized them in their separated positions by eiectrophoresing alkaline cyanoborohydride into the suspending medium. When used with small disposable columns this zonal imnobilization and direct staining procedure provides a speedy and inexpensive alternative to fraction collecting. Further, it has been found that the dissociable complexes that are formed by reversible interaction of fibrin monomer with fibrinogen are not separable from fibrinogen under conditions commonly used for gel filtration. That is because solutlity of fully polymerizable fibrin depends on its interaction with fibrinogen. To detect such fibrin in plasma, we equilibrate columns with buffer containing rabbit fibrinogen as a carrier to maintain solubility. Studies on patients indicate that the stability of fibrin complexesin blood may vary in characteristic way depending on underlying disease processes. (Supported by LISPHS grants HL-16361 and HL-19767).
Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment
Authors: Kudryk B. , Blombäck M.
Published: May 2019

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.
A Hitherto Undescribed Naturally Occurring Plasma Antagonist of Activated Factor X Inhibitor (Antithrombin III)
Authors: Yin E.T. , Salsgiver W.J. , Tangen O.
Published: May 2019

Circumstantial evidence suggested that normal human plasma contained a substance regulating the neutralization of F. Xa by F. Xa inhibitor (XaI), (Yin et.al., Adv. Exper. Med.& Biol., 52: 239, 1975, Plenum Press, N.Y.).

This plasma component has now been isolated and partially purified in our laboratory, and tentatively designated as “Anti-Xal”.

In experiments employing purified components, when Anti-Xal was incubated at 37°C with F. Xa. Xal and heparin for two minutes at pH7.5, the amount of F. Xa inhibited was inversely proportional to the Anti-XaI concentration. But, when the F. Xa was replaced by thrombin in the incubation mixture, the neutralization of thrombin clotting activity was undisturbed. Anti-Xa I was found to be neither PF3 nor PF6.

These and other data strongly suggest that the “Antithrombin ill pathway” is more complex than currently believed to be. In circulating blood an equilibrium state must exist between Anti-Xal and Xal. Under certain conditions when the Anti-Xal activity is predominant the rate of F.Xa neutralization by Xal then becomes slower than the activation of prothrombin to thrombin by F.Xa.
Antithrombin III, Heparin Cofactor and Antifactor Xa in Relation to Age, sex And Pathological Conditions
Authors: Panicucci F. , Sacripanti A. , Pinori E. , Vispi M. , Conte B. , Lecchini L.
Published: May 2019

Determinations of AT-III activity, heparin cofactor activity, antifactor Xa activity and AT-III protein were carried out in 200 healthy adults, evenly distributed within age and sex groups, in 60 patients with cerebral thrombosis and in 20 oral contraceptive users. There was a positive correlation between AT-III protein and its activitiesin normal subjects and in patients with cerebral thrombosis. In oral contraceptive users the positive correlation was between AT-III protein and its activities, antifactor Xa activity excepted. The mean AT-III protein and heparin cofactor activity values decreased in males with age and were significantly lower in the groups between 50 and 70 years. The mean AT-III protein and heparin cofactor activity values decreased slightly in women in fertile age and were lower in the 40 to 50 age-group. The mean AT-III protein and its activities values did not show any variation in the patients with cerebral thrombosis. The mean antifactor Xa activity value in the women, taking the pill for 3 months, decreased, whereas the other AT-III activities and AT-III protein were unchanged.