bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromogenic Substrate Methods For The Determination Of FVIIIc, Endotoxin And Plasminogen Activator
Authors: Eriksson E , Rosén S , Knös M , Friberger P
Published: July 2018

FVIII clotting activity in plasma and concentrates can be assayed with good reproducibility in the range 20-120% (C.V. approximately 3%) using purified FIXfl and FX in excess. By incubating the sample with above factors, Ca2+ and phospholipid, FX is activated in proportion to the amount of FVIIIc. Generated FXa is measured with the substrate S-2222. FVIIIc in the range below 20% can be determined by modifying the reaction conditions.

Endotoxin in water solutions can be accurately determined down to 1 pg/ml by incubating the sample with an excess of Limulus lysate. The generated enzymatic activity which is linear in proportion to the amount of endotoxin, is then measured with the substrate S-2423 (Ac-Ile-Glu-Gly-Arg-pNA). Accurate endotoxin determinations in plasma and cerebrospinal fluid are also possible after destroying interfering inhibitors by heat treatment.

Plasminogen activator activities can be measured via plasminogen and the plasmin substrate S-2251 or directly by substrate S-2288 (H-D-Ile-Pro-Arg-pNA). Interfering activities can be minimized by strategic selection of various protease inhibitors.

In all the methods the assay conditions, reagents and procedure have been optimized.
Effect Of Prostacyclin (PGI2) On The Release Of β Thrombo- Globulin (βTG) And Platelet Factor 4 (PF 4) During Extracorporeal Circulation (ECC)
Authors: Matthias F R , Ditter H , Heinrich D , Simon J , Söhngen D , Schleussner E , Walter P
Published: July 2018

A prospective and double-blind study was conducted on 40 patients with coronary bypass operation. In 20 patients 8 ng of PGI2 per min and kg body-weight were infused during ECC. The amount of heparin administered per kg was the same in both groups. Drop in platelet count during ECC however was less and bleeding times were longer under PGI2 infusion. βTG was released from platelets immediately after onset of ECC and remained at a nearly constant level. PF 4 concentration in plasma increased continuously over the whole period of ECC (1600 ng/ml or 1200 ng/ml, max. values). PGI2 infusion leads to a significant reduction of the amount of both platelet constituents released (950 ng/ml or 750 ng/ml, max. values). After ECC βTG and PF 4 dropped down immediately to preoperative values. No differences in per-/ postoperative blood loss and in the amount of heparin bound to patients' antithrombin III could be observed.
Central Venous Catheters And Thrombotic Complications. A Comparison Of Three Different Catheter Materials
Authors: Lindblad B , Efsing H O , Mark J , Wolff T
Published: July 2018

Different frequences of thrombosis due to central venous catheters (CVC) is reported. Only few studies have compared the thrombogenecity of different catheter materials. This study was undertaken to evaluate and compare the frequency of thrombotic complications with three catheter materials.

MATERIAL: The patients were randomized into three groups receiving: teflon catheter, siliconized polyethylen. catheter and heparinized polyethylen catheter.

METHODS: Percutanous insertion (subclavian or internal jugular vein) was made under strict aseptic conditions as well as the daily care of the CVC. The condition of the veins was assessed by either phlebography or post-mortem examination. Phlebography was usually made on the seventh day after insertion of the CVC by contrast injection in a cubital vein.

RESULTS: Of 110 patients 99 were examined by phlebography and 20 by post-mortem.

Repeat phlebography was made after 3 to 4 weeks use of CVC in 14 patients and showed no late thrombotic development. Two patients had pulmonary embolism, one with fatal outcome. No other source for embolism than the thrombosis around the CVC was found.

CONCLUSION: The siliconized polyethylencatheter had a significantly higher frequency of thrombosis than teflon or heparinized polyethylen catheters had. CVC did not give any late thrombotic development. Two patients developed pulmonary embolism, one with fatal outcome.
Evaluation Of The Thrombogenicity Of Four Dacron Arterial Grafts In An Artificial Circulation
Authors: Deacon P , Donaldson D R , Crow M J , Rajah S M , Kester R C
Published: July 2018

A major consideration in the choice of vascular prosthesis is the thrombogenic potential of its luminal surface. As platelets play a significant role in arterial thrombosis we studied graft-platelet interaction ‘in vitro' with an artificial circuit as a means of comparing the thrombogenicity of grafts. The types of grafts evaluated were, a plain knitted (Meadox ‘Cooley'), the pyrolitic carbon coated knitted (Meadox ‘Biolite', a double velour (Meadox ‘Microvel’ and a filamentous double velour (USCI ‘Sauvage’ The artificial circulation comprised twin loops of silicone tubing, one a test circuit containing a 15 cm × 8 mm diameter graft, the other a control circuit. Both circuits were perfused simultaneously with fresh heparinised blood from the same donor, for 60 mins at 37°C, with a pressure 120/80 mm Hg and a flow 140 mls/min. Six samples of each graft were tested. Thrombogenicity of the graft was assessed by the change in platelet count, adhesion and aggregation measured at 0, 15, 30 and 60 minutes of perfusion. The luminal surface was examined at the end of perfusion, by Scanning Electron Microscopy (SEM), and counts of adherent platelets made. There was a fall in platelet count, adhesion and aggregation in the test circuits during perfusion, this fall being similar for each type of graft, except the filamentous double velour where the fall in platelet count and adhesion was marginally greater. Serial counts of platelets adherent to the luminal surface (SEM) showed three grafts to be very similar (Biolite 84 ± 24, Cooley 2 111 ± 15 and Microvel 126 ± 37 respectively). The Sauvage graft had a very significantly higher number of adherent platelets (p<0.001) with a count of 490 ± 80.

The results of this study indicate that the filamentous double velour has a more thrombogenic surface than the other grafts, when evaluated in our experimental model.
Changes In Arachidonate-Containing Complex Lipid During Platelet Stimulation
Authors: Rittenhouse-Simmons S
Published: July 2018

We have examined changes in the 3H-arachidonic acid (3HC20:4) content of phospholipid following stimulation of human platelets by thrombin, ionophore A23187, or collagen. Resolution of lipids and lipid metabolites was achieved using HPLC and thin-layer chromatography after extraction of platelets by a modified Bligh and Dyer technique. The most rapid changes, leading to formation of TXB2, occur in response to thrombin and A23187. Both stimuli induce marked losses of 3HC20:4 from phosphatidylcholine (PC) and phosphatidylinositol (PI) within 30 sec. Collagen also induces losses in PI and PC and formation of diglyceride (DG), which become appreciable only after 1-3 min. Whereas thrombin and A23187 exhibit similarities in terms of the rate of response, these agents differ with regard to the phospholipases activated. Exposure of platelets to A23187 causes some activation of Ca+2-dependent PI-specific phospholipase C (PI-PLC); however, A23187 is an inefficient stimulus for this enzyme. Ionophore induces the formation of 1/4 to 1/6 as much PI- PLC-derived DG as does thrombin when comparable amounts of PI are hydrolyzed. A similar discrepancy is found with respect to generation of phosphatidic acid. Simultaneous addition of A23187 and thrombin does not impair the formation and metabolism of DG promoted by thrombin alone. Further, whereas indcmethacin (1-100 μg/ml) exerts no inhibitory effects on PLC or the formation of DG in stimulated platelets, indomethacin does inhibit 1) the loss of 3HC20:4 from PC in response to thrombin and 2) the loss of 3HC20:4 from PC and PI in response to A23187. Indomethacin has been reported to inhibit platelet phospholipase A2. It is clear from these studies that the activation of PI-PLC is not triggered merely by a general Ca+2 flux. In contrast, a shift in platelet Ca+2 stores appears to be a sufficient stimulus for phospholipase A.
Endothelial Cell-Lined Vascular Prostheses
Authors: Burkel W
Published: July 2018

While considerable effort has been expended to develop blood compatible artificial devices, the only truly non- thrombogenic surface known is that of vascular endothelium. Early prosthetic studies emphasized graft material from the surgical viewpoint. Later the emphasis changed to the physical configuration of the graft (woven, knitted, velour, porous, nonporous grafts, etc.) which gave the best tissue ingrowth, stable flow surface and hopefully an endothelial lining. Since vascular grafts seldom become lined with endothelium in man as a natural consequence of the healing process, methods to enhance the development of an endothelial surface would be of considerable value in reducing thrombosis, infection and the destruction of blood elements. To accomplish this objective, a wide variety of cells and tissue fragments have been added to grafts in order to produce pseudointimas.

In our experiments endothelial cells have been isolated from the external jugular veins of 20-30kg foxhounds using trypsin and collagenase. The harvested cells were either cultivated in vitro for 14 days and then seeded into Dacron and PTFE vascular grafts during preclotting, or seeded into grafts immediately after derivation. Grafts were implanted as thoracoabdominal bypasses (25-30 cm in length) and incorporation of the graft and the formation of an endothelial lining followed from 1 day to 1 year. Grafts treated in an identical manner, but preclotted without endothelial cells served as controls.

Endothelial cells are first visible in seeded grafts at 4 days, approach 70% coverage of the graft surface by 2 weeks and 90% at 1 month. Four month seeded grafts are completely lined with endothelium, while unseeded grafts still have 20-30% of their surface covered with a fibrin coagulum at 6 months. Preliminary results indicate that blood parameters are greatly perturbed initially, but return to normal levels with the evolution of an endothelial lining in seeded grafts.
Epidemiology Of Myocardial Infarction: Magnitude Of The Problem, Cause And Risk Factors
Authors: Kannel W B
Published: July 2018

Coronary heart disease is a common, highly lethal, disease which frequently attacks without warning and too often presents with sudden death as the first symptom. Chances of an American male developing CHD before age 60 are one in five.

Most angina, infarctions and sudden deaths represent medical failures which should have been forecasted and prevented. About 30% of first MI's will shortly develop angina and experience a per annum death rate, half of which will be sudden deaths. Reinfarctions will occur at 6% per year and half the recurrences will be fatal.

No major innovations are needed to identify coronary candidates or to establish their risk from the joint effect of known risk factors. However, all have much to learn about motivating changes in behavior required to control the major risk factors such as cigarette smoking, faulty diet, overweight, sedentary living, abnormal lipids, hypertension and impaired glucose tolerance.

Low density lipoprotein cholesterol promotes atherogenesis whereas HDL-cholesterol is protective, and the net effect is judged by their ratio. Hypertension, systolic or diastolic, labile or fixed, at any age in either sex is a powerful contributor to CHD. The impact of diabetes is greater for women, diminishes with age and varies depending on coexisting risk factors.

Optimal risk evaluation requires quantitative combination of risk factors so as to include persons with multiple marginal risk factor abnormalities who are at high risk.
Large (But Simple) Trials: When Are They Justified
Authors: Meinert C L
Published: July 2018

Trials, designed to evaluate the therapeutic or prophylactic effect of a treatment on a chronic condition such as heart disease, often require thousands of patients to provide the power required to detect a small but clinically important treatment effect. The complexity and cost of the large scale multicenter trial has made it difficult to obtain the support needed to carry them out. There is a need to find ways to reduce the cost of large scale trials if they are to remain in our armamentarium of evaluation tools, especially in this age of shrinking research budgets. This paper examines the major factors which contribute to the cost of large scale trials and conditions required before it is possible to consider simplified data collection and follow-up procedures. Special emphasis is given to the difficulty scaled down data collection efforts may cause when the safety of the study treatment is in doubt and when the test treatment must be administered over an extended period of time, as in drug trials providing long term therapy for a chronic condition.
Prospects For Chromogenic And Fluorogenic Substrate Use In Biochemistry And Blood Protease Assay
Authors: Jackson Craiq M
Published: July 2018

Kinetic parameters describing the hydrolysis of peptide p-nitroanilide substrates by thrombin, Factor Xa, and activated Protein C indicate that a high degree of selectivity for each of these proteases can be achieved by using appropriate substrates. Determination of kcat (the maximum velocity per mole of enzyme) for peptide p-nitroanilide substrates indicates that sensitivity sufficient to detect pM concentrations of these proteases can be obtained. The use of fluorogenic substrates should increase sensitivity, although the absence of data for maximum velocities of hydrolysis of peptide fluorogenic substrates precludes quantitative statements about the extent of increase in sensitivity that may actually be obtained. The use of more than one substrate provides an opportunity to selectively assay individual enzymes present in a mixture of proteases. The selectivity of the various assays can be enhanced by use of competitive inhibitors such as those developed in several laboratories for use as potential antithrombotic agents. Although manual methods may be too tedious to be practical in complex situations, the automated methods which handle multiple samples can make such multiple-substrate based assay methods practical. The fact that a single function of the protease coagulation factors, namely the peptide bond hydrolysis function is being assessed by peptide chromogenic and fluorogenic substrates will permit more specific information to be obtained about these multifunctional molecules.
The Effect Of Thrombin On The Electrophoretic Mobility And Functional Activity Of Various Factor VIII Molecular Forms
Authors: Seghatchian M J , Mackie I I
Published: July 2018

Several distinct populations of factor VIII procoagulant activity (VIIIC) were observed in some clinical concentrates after agarose gel electrophoresis. It is not yet known whether these different forms are the products of proteolytic digestion, or subtle conformational changes of FVIII structure, which may occur during concentrate preparation. Low thrombin concentrations activate FVIII in a time/concentration dependent manner, and the 2 proteins have high affinities for one another. We have studied the properties of the various FVIII forms before and after incubation of concentrate with highly purified α-thrombin. Agarose gel electrophoresis was used to monitor: a) quantitative changes in functional activity (by 2 stage clotting (VIII:C) and amidolytic (VIII: CAm) assays); b) the binding of 125I- anti VIII:C (*IgG) to FVIII: C; c) the crossed immunoelec- trophoretic (CIE) pattern of FVIII: RAg.

In the absence of thrombin, FVIII concentrate showed 2 main populations of FVIII: C and VIII: CAm. The radioactivity (VIII: CAg) pattern correlated with the slower moving of these populations. Low thrombin concentrations (0.0001-0.01 u/nl) caused no detectable change in FVIII: C level in the slow peak but additional peaks of activity appeared at the sample well, and between the well and slow peak. This was accompanied by a decrease in the fast moving peak, and the appearance of a third new peak with slightly less mobility. Higher thrombin concentrations (0.5u/ml) caused a marked decrease in the slow moving peak, with a concomitant increase in the well peak, by all methods. Increasing thrombin concentration progressively reduced *IgG binding, and a pattern similar to serum was obtained. Thrombin caused a slightly faster migration of FVIII: RAg, a well peak was sometimes seen. Thrombin therefore appeared to act preferentially on the lower molecular weight FVIII activity, and the high molecular weight peaks that occurred may represent complexes of FVIII with thrombin and/or other contaminant proteins present in concentrate such as fibrinogen or fibronectin.
Factor VIII Is A Calcium-Binding Protein
Authors: Cruickshank W H , Tackaberry E S , Palmer D S , Rock G A
Published: July 2018

The high molecular weight complex of Factor VIII is known to dissociate into two subunits when chromatographed in a high ionic strength solution containing either calcium or sodium salts. Calcium also has an essential role in maintaining Factor VIII activity during storage (AABB 1980). These two findings suggest ah integral involvement of calcium in the basic structure-function relationship of the Factor VIII molecule. Consequently, we have examined the binding of 45Ca to both the low molecular weight (LMW) procoagulant subunit of Factor VIII and the high molecular weight (HMW) complex using PAGE and column chromatography on Sepharose 4B. When the HMW complex of Factor VIII was isolated from cryoprecipitate by standard chromatographic procedures, incubated with 45CaCl2 and subjected to polyacrylamide gel electrophoresis, the resultant autoradiogram demonstrated that all of the 45Ca was associated with the HMW material which did not enter the gel. When this HMW Factor VIII was incubated with 45Ca and then dissociated by column chromatography in a buffer containing 0.25 M CaCl2, all of the 45Ca was associated with the material eluting in the 2.3 Vo region. This corresponds to the elution volume of the LMW, procoagulant subunit of Factor VIII. Dialysis against buffer was not effective in removing the 45Ca. The data indicate that calcium is tightly associated with the low molecular weight subunit of Factor VIII and presents further corroborative evidence that calcium has an essential role in determining both the chemical and physical properties of the Factor VIII molecule.
Oral Bleeding In Hemophilia; A 21 Year Composite
Authors: Sonis A , Andes W A , Smith B
Published: July 2018

The charts of 132 patients with proven factor VIII deficiency were analyzed for severity of disease, mean age, mean age at diagnosis, and number of patients diagnosed following a bleeding episode. In addition, the frequency of bleeding episodes requiring factor infusion was determined for a single calendar year. Infusions for dental care (i.e. restorations, extractions) were excluded from the study. Severe and moderate hemophiliacs comprised 71% of the patient’s studied. Their mean age was 27 years and their mean age when a bleeding disorder was suspected was under 1 month. Mild hemophiliacs comprised 27% of the patients studied. Their mean age was 27 years and their mean age when a bleeding disorder was suspected was 8 months. Persistent oral bleeding resulted in the diagnosis of hemophilia in 9% of the patients. None of these patients were severe hemophiliacs. The group diagnosed as a result of persistent oral bleeding was comprised of 11 mild and 1 moderate hemophiliacs. This constituted 29% of all the mild hemophiliacs and 2% of all the moderate hemophiliacs. The average annual frequency of bleeding episodes per patient requiring factor was as follows; severe-24 infusions of which were for oral bleeds, moderate-21 infusion of which were for oral bleeds, mild-16 infusions for which were for oral bleeds. The anatomical distribution of oral bleeds requiring infusion revealed the lips most frequently involved (48.5%) followed by the tongue (35.3%), buccal mucosa (15.4%), gingiva and palate (0.9%). The results of this study suggest that mild hemophiliacs are diagnosed at a later age than moderate and severe hemophiliacs, and that 29% of mild hemophiliacs are diagnosed as the direct result of an oral bleed. In view of these findings, a persistent oral bleed should alert the physician or dentist to a possible bleeding disorder.
An Enzyme-Linked Immunosorbent Assay (ELISA) For Factor VIII Inhibitors
Authors: Miller C H , Hilgartner M W , Lopez D R
Published: July 2018

ELISA microtechniques are suitable for measurement of a variety of antigens and antibodies. They are sensitive, safe, and easy to perform. Enzyme-labeled antibodies to factor VIII and factor IX can be used to measure antigen in a “sandwich” assay analogous to the immunoradiometric assay (IRMA). Likewise, specific antibodies can be easily quantitated in plasma samples. Quantitation of F. IX inhibitors by ELISA has been performed by Orstavik and colleagues. We have studied use of the ELISA system for measurement of inhibitors to factor VIII. Purified factor VIII prepared from concentrate is bound to plastic microtiter plates, which are then incubated with plasma dilutions. After washing to remove non-bound material, heterologous anti-human IgG labeled with alkaline phosphatase is added. It binds to any hyman IgG remaining on the plate. Following incubation and washing, the enzyme substrate, p-nitro- phenyl phosphate, is added. Degradation of the substrate produces a color change which is read spectrophoto-metrically. A “through-the-plate” reader allows rapid OD measurements without transfer of the well contents. Ten or more samples in dilution series can be tested simultaneously. The inhibitor titer may be quantitated by comparison with a standard inhibitor measured in Bethesda units by a clotting assay or by direct comparison with the OD curve produced by known quantities of human IgG. Twelve known inhibitors from hemophilia patients and two acquired non-hemophilic inhibitors showed good correlation with the Bethesda unit method. Five normal individuals and five hemophiliacs with no history of inhibitor by clotting assay showed no anti-IX inhibitor by this test.
Inhibitory Effects Of Albumin On Ristocetin-Induced Fibrinogen Precipitation
Authors: Zuhlke J A , Mattson J C , Bell T G
Published: July 2018

It has been noted that ristocetin causes the precipitation of fibrinogen. In ristocetin cofactor assays, this precipitation can mask the platelet agglutination endpoint. At higher concentrations of ristocetin (>1.5 mg/ml) a heavier precipitate forms. Bovine albumin when added to the test system will inhibit this precipitation, von Willebrand’s Disease (vWD) plasma was tested with varying concentrations of ristocetin and albumin. When 0, 10.1 and 13.6 mg/ml of albumin were used in the ristocetin cofactor assay (ristocetin at 1.2 mg/ml) rapid endpoints were achieved and levels of ristocetin cofactor for vWD plasma were not in the expected abnormal range. Albumin concentrations of 20 mg/ml produced low ristocetin cofactor levels for vWD plasma and normal levies for pooled normal plasma. Concentrations of bovine albumin greater than 30 mg/ml made endpoints difficult to see and greatly prolonged the time to reach the endpoint. Since higher ristocetin concentrations precipitate more fibrinogen, a studying varying ristocetin concentration was also undertaken. When 20 mg/ml of albumin was used, ristocetin in concentrations of 1.0-1.2 mg/ml produced normal activity for pooled normal plasma but low ristocetin cofactor activity for vWD plasma. Ristocetin in concentrations greater than 1.5 mg/ml resulted in normal ristocetin cofactor activity for the vWD plasma. Ristocetin at 0.5 mg/ml did not induce platelet agglutination that was detectable in this assay. Similar studies were performed using canine plasma and it was ascertained that 1.0 mg/ml of ristocetin must be used with this species because higher concentrations of ristocetin (>1.2 mg/ml) produce normal results in vWD plasma. The albumin effect in the canine assay is similar to that found in the human ristocetin assay. These data suggest that it is imperative to standardize albumin and ristocetin concentrations in ristocetin cofactor assays.
Effect Of DDAVP In Patients With Von Willebrand’s Disease And Hemophilia A
Authors: Warrier A I , Hillman C , Lusher J M
Published: July 2018

European investigators have reported on the efficacy of l-deamino-9-D-arginine vasopressin (DDAVP) in von Willebrand’s disease (vWD) and mild hemophilia A. We have thus evaluated the effects of a single intranasal dose of DDAVP (200ugm of the more dilute form available in the U.S.), in 12 individuals with vWD and in 4 with moderate hemophilia A. Crossed immunoelectrophoresis of VIII: RAg demonstrated normal electrophoretic mobility in each of the vWD subjects. Components of the factor VIII system (VIII: C, VIII: RAg, VIII: R Cof.) were assayed pre - and 90 and 180 minutes post- DDAVP. Each of 11 subjects with mild or moderate vWD had an increase in VIII: C activity (avg. 2X increase), 8 of 11 had an increase in VIII: R Cof, and 9 of 11 had an increase in VIII: RAg. The twelfth vWD subject, who had severe vWD, had no rise in any of these components. Of 4 vWD subjects who had pre- and post-DDAVP template bleeding times (BT) performed, the only one who had a prolonged baseline BT showed a normal BT 90 minutes post-DDAVP. One vWD subject, in whom we had documented an increase in all F. VIII components after DDAVP, later underwent dental extractions 90 minutes after DDAVP. No excessive bleeding was noted. Four individuals with moderate hemophilia A (baseline VIII: C values of 0.02-0.10 u/ml) were also studied. Three had a rise in all components of the factor VIII system post-DDAVP while the fourth did not. No undesirable side effects were noted in any of the 16 subjects who received DDAVP. We conclude that even the more dilute form of DDAVP available in the U.S., when given intranasally, results in transient improvement in selected individuals with vWD or moderate hemophilia A. This drug thus warrants further study as an alternative to blood components in the management of vWD, as well as in mild and moderate hemophilia A.
Factor VIII Antigen And Venostasis Test In Diabetes Mellitus. A Possible Useful Tool To Detect A Latent Endothelial Suffering
Authors: Giustolisi R , Musso R , Lombardo T , Russo M , Cacciola E
Published: July 2018

In the mature onset Diabetes Mellitus (DM) high levels of Factor VIII antigen (FVIII R: AG) were reported (Bensoussan et al., 1975, Diabetologia 11, 307; Lufkin et al., 1979, Metabolism 28, 63). The elevated FVIII R: AG levels observed in DM, as well as in other vascular diseases, were claimed as an index of endothelium damage (Boneu et al., 1975, Lancet 1, 1430; Corda et al., 1979, Thrombos. Res. 14, 805). Moreover, since the FVIII R:AG is involved in platelet adhesion to s b- endothelium, its increase might have prognostic significance as thrombogenic risk factor. But, in DM plasma FVIII R: AG levels could be also within normal range, so that the significance as index of endothelial suffering in such evenience would be lacked. Here we report evidence that in DM without clinical signs of vasculopathy, even if the FVIII R: AG plasma levels are in the normal range, the existence of endothelial suffering could be likewise detectable by the simple ve nostasis test, which, notoriously, represents a damaging endothelium stimulus. To test such a possibility we investigated the behaviour of plasma FVIII R: AG levels after venostasis (performed at forearm pressure of 10 mmHg over diastolic × 20 min) in 12 diabetic patients without overt signs of vasculopathy and exhibiting FVIII R: AG in normal range. This group was matched with 10 normal subjects of comparable sex and age. Measurements of the serum LDH levels before and after venostasis served to prove cellular damage when they increased more than 50%. The diabetic patients showed after venostasis an increase of FVIII R: AG (Laurell’s method)which was significantly higher than normal controls (P<0.005). Therefore it might be assumed that in diabetic subjects a latent vascular endothelium suffering, even without overt signs of vasculopathy, could exist and it could be detectable by venostasis test.
The Functional Heterogeneity Of Clinical FVIII Concentrates
Authors: Mackie I J , Seghatchian M J
Published: July 2018

There is an increasing tendency to use more highly purified FVIII preparations, and more sophisticated fractionation methods. In some cases new populations of FVIII:RAg have been produced that have a greater or lesser physiological activity. The characterisation of clinical concentrates on the basis of their biological activity is therefore extremely important in the search for more effective therapy.

We have investigated the activity of several high purity concentrates (Hemofil, Koate, Actif-VIII) as well as low purity pooled cryoprecipitate, with and without added 125I- anti-VIII:C(*IgG or *FAB), after electrophoresis in 1% agarose gels.

In concentrates with a high FVIII:RAg concentration(25-30Iu/ml), 2 or more FVIII:RAg peaks were seen, with partial or complete identity. The main one migrated in the α 2 region, but a more anodal peak was frequently seen. The relative concentration of fast moving VIII:RAg in cryoprecipitate was too low to allow detection by immunoelectrophoresis. In the presence of *IgG or *FAB, only one main peak of radiolabelled VIII:C was noted, which corresponded with the larger molecular forms of VIII:RAg. In contrast, bioassays based on FXa generation (VIII:C and VIII:CAm), revealed 2 main peaks of activity in concentrates, cryoprecipitate and plasma. The slower moving peak correlated well with the radiolabelled VIII:C pattern. The fast moving peak in concentrate corresponded with the trailing edge of the fast moving VIII:RAg peak. This activity was not neutralised by increasing the *IgG level, or addition of anti-IXc IgG to the sample. No enzymic activity was detected in this region using a wide range of chromogenic substrates. However, Al(OH)3 adsorption did remove the activity. These forms of FVIII may represent a native subpopulation or proteolytic breakdown product, which has lost some antigenic determinants, but retained biological activity. This material, which has a lower affinity for anti VIII:C and RAg, could be present in plasma and be selectively concentrated during fractionation procedures.
Heated Lyophilized Factor VIII Concentrate - Additional Preliminary In Vitro Studies
Authors: Rubinstein A
Published: July 2018

The possibility that heating of lyophilized factor VIII in a water bath at 60°C for 10 hours will not be sufficient for significant inactivation of hepatitis virus in the lyophilized powder led to different heating experiments. Heating of lyophilized factor VIII at 100°C for 30 minutes led to severe deep brown discoloration and this experiment was abandoned. Heating of lyophilized factor VIII powder for 16 hours at temperatures between 62-64°C followed by storage at 6°C for 4 weeks still resulted in greater than 80% recovery of factor VIII as compared to an unheated control, absorbance of the reconstituted factor VIII at 580 nm resulted in .127 for the heated reconstituted factor VIII and .117 for the unheated control; immunoelectropheresis against human serum showed overall increase in anodal migration as compared to an unheated control. Clearly it must be determined with future chimpanzee studies which heating temperature and length of time is necessary for significant inactivation of hepatitis virus.
Asynchronous Generation Of Kinin And Kallikrein Activity Following Soluble Activation Of Plasma
Authors: Bubnic A M , Zuffi T R , Fournel M A
Published: July 2018

In an effort to assess the physiological significance of a soluble activator of Factor XII, the kinetics of F. XII and prekallikrein activation and subsequent kinin formation have been investigated using dextran sulfate. Employing an amidolytic assay for kallikrein and a bioassay for kinin activity a reproducible lag in the generation of detectable kallikrein activity has been demonstrated in the presence of explosive kinin generation. Time, temperature and concentration kinetics have been determined as well as characterization of this system with prekallikrein activator (PKA; B-XIIa; XIIf), selective inhibitors and deficient plasmas. These studies have shown that kinin activity is generated in substantial amounts prior to the generation of any significant kallikrein activity by the activation of F. XII, suggesting that the physiological role of prekallikrein activation in kinin generation may be relatively minor. In vivo animal studies with PKA appear to confirm this finding with insignificant alterations in prekallikrein titers in the presence of marked systemic kinin-mediated hypotension.
The Factor XII-Independent Plasminogen Proactivator System Of Plasma Includes Urokinase-Related Activity
Authors: Kluft C , Wijngaards G , Jie A F H
Published: July 2018

Antibodies to urokinase have been shown to quench part of the fibrinolytic activity of plasma, not involving the extrinsic system. Previously, the intrinsic system has been subdivided into two parts, one dependent and another independent of factor XII, for activation. Both represent approximately 50 BAU activator activity/ml plasma. The aim of this study was to further locate the UK-related activity within the intrinsic fibrinolytic pathways and to delineate its participation in fibrinolysis tests in vitro.

Antibodies to UK (AUK) were shown not to quench activities of contact factors such as factor XII, prekallikrein and factor XI in coagulation and chromogenic substrate assays. AUK inhibited in normal plasma, Hageman trait and Fletcher trait plasma a discrete portion of approximately 50 BAU activator activity/ml, apparently the activator activity derived from the factor XII-independent system.

Immunoadsorption of plasma with immobilized AUK resulted in a depletion of approximately 50 BAU activator activity/- ml. The contact factors were undisturbed, as were plasminogen and the extrinsic activator. Additions of UK did not restore lost activity, suggesting that AUK has removed the proactivator component.

The amount of UK-related activator activity compares to approximately 0.4 IU urokinase activity/ml, and shows a fairly constant level in individuals and is not enhanced by known stimuli for extrinsic activator release.

Significant contributions to blood fibrinolytic activity in current assay systems, including the dilute blood clot lysis time method could be demonstrated.
Evaluation Of The Plasma Kallikrein System In Argentine Haemorrhagic Fever (FHA)
Authors: Giavedoni E , Molinas F C , Maiztegui J I
Published: July 2018

Previous work in this laboratory have demonstrated that the coagulation and complement systems are involved in Argentine haemorrhagic fever (AHF). Due to their interrelation with the plasma kallikrein system we have decided to evaluate the latter measuring Factor XII (F XII), prekallikrein (PK) and kallikrein inhibitors (KIs) in 18 patients with AHF. The patients were grouped according to clinical parameters in the following forms: mild (15 patients), moderate (2 patients) and severe (1 patient). Values of F XII, determined with F XII deficient plasma, were within normal range. The concentrations of PK and KIs (enzymatic assay) in AHF patients were respectively: days 5-7, 106.9 uM/ml/h, 0.99 U; days 8-10, 97.4 uM/ml/h, 0.98 U; and days 11-12, 104.8 uM/ml/h, 0.92 U. These levels were similar to our normal controls, however the 2 patients with the moderate clinical form presented on days 9-11 values lower than normals (PK 58.6 uM/ml/h; KIs 0.82 U). Simultaneously we investigated the lengthening of the partial thromboplastin time activated with kaolin (PTTK) observed in these patients. In order to demonstrate the presence of an early phase inhibitor we determined the PTTK using mixtures of patient and normal plasmas incubated for 20 min at 37°C. The results obtained discarded the presence of such an inhibitor. Our study indicate that there is no activation of the plasma kallikrein system in this group of patients. Further studies are needed with more cases of the moderate and severe clinical forms of AHF.
Evaluation Of Contact Activation Of The Intrinsic Coagulation System In Evacuated Tubes: Chromogenic Substrate Assay Equivalent To The Non-Activated Partial Thromboplastin Time
Authors: Edlefson D S , Aiyappa P A
Published: July 2018

Plasma was collected and stored in plastic tubes and commercially available blood collection tubes to study the effect of the type of surface on contact activation. To determine if activation had occurred, a sensitive chromogenic substrate assay was used. A non-activated partial thromboplastin reagent was employed with a synthetic tripeptide chromogenic substrate to measure the thrombin activity. The results indicated that the evacuated tubes showed substantial contact activation as evidenced by the increased levels of thrombin generated, compared to the plasma stored in plastic. Total siliconization of the glass surface eliminated contact activation. The chromogenic substrate assay equivalent to the non-activated Partial Thromboplastin Time offers a sensitive, easy and faster method for studying contact activation.
The Hormone - Dependent Concentration Changes Of Factor XIII (Fibrinoligase) During Pregnancy
Authors: Ebert Ch , Berg H , Zimmermann R E
Published: July 2018

The effects of hormones on blood coagulation and fibrinolysis have been studied extensively since oestrogen / progesterone preparations were used for contraceptive purposes. There is a general agreement that such preparations cause a rise in plasma levels of fibrinogen, prothrombin and factors VII, IX, and X. In Comparison, most of the coagulation factors synthesized by the liver show a gradual increase during normal pregnancy. Only factor XIII and calcium reveal a tendency to decrease. In 90 patients out of a normal population the plasmatic factor XIII concentration declined from 0.39 units in the first trimenon to 0.29 and 0.23 units in the second and third trimenon. Since the fibrinogen concentration is higher than during normal menstrual cycles the reduction of factor XIII may be caused by a decreased synthesis or an increased clearance rate from circulation. The abnormal haemostatic conditions during gestation could be interpreted as a physiological response to the increase of oestrogen and progesterone leading to a hypercoagulative state. Reduced levels of factor XIII may counterbalance the increased rate of fibrin formation and serve as protection against thromboembolic complications.
Factor XIII, Tissue Transamidase And Malignancy
Authors: Zuch A , Kloczko J , Bielawiec M , Buluk K , Pilecka E
Published: July 2018

The aim of the investigation was to compare the activity of factor XIII in patients with malignant neoplasma with the changes in the blood plasma of rats with Guering epitheliomas and to evaluate the effects of this tumour on the activity of tissue transamidase capable of stabilizing fibrin.

The investigations were carried out on 125 patients with several neoplasmas and 50 healthy subjects. A significant decrease in f. XIII activity in plasma was found in these patients, as well as decrease in the concentration of free SH groups on which its activity depends. Similar changes In the blood plasma were noted in rats examined 30 days after transplantation of the Guerin tumor. In the organs of these rats a fall in transamidase activity was observed. On the other hand the tumor rich in SH groups contains a very large amount of enzyme which like f. XIII is capable converting fibrin polimer into cross-linking fibrin. Concentration of this transamidase in neoplastic tissue was significant higher than in the tissue of the control animals.

Our results suggest that activity of transamidase and SH groups are connected with neoplastic process and that decrease of f. XIII activity in the blood plasma is the result of its consumption during intravascular coagulation initiated by neoplasma.
The Effect Of The Local Anaesthetic Tetracaine On Prothrombin Activation
Authors: Orthner Carolyn L , Kosow David P
Published: July 2018

The conversion of prothrombin (Pro) to thrombin by the serine protease Factor Xa, an important reaction in blood coagulation, is thought to occur on a negatively-charged phospholipid (PL) surface. Kinetic studies using purified human proteins and PL vesicles (25% phosphatidylserine, 75% phosphatidylethanolamine) at pH 7.5 in the presence of 5mM CaCl2 (Ca) showed that mMol tetracaine (4-(butylamino) benzoic acid 2-(dimethylamino)ethylester) (Tcn) inhibited Pro activation. The rate of Pro activation as a function of Ca concentration was positively cooperative (Hill coefficient = 2.5) in the absence or presence of Ten (3mM) while the V was decreased 35% indicating Ten was not simply displacing Ca. When PL was varied at several constant levels of Ten, noncompetitive inhibition was found with a Ki of 1.8mM. The fluorescent probe l,6-diphenyl-l,3,5-hexatriene (DPH) was incorporated into PL. Tcn increased the fluorescence polarization of DPH with a 71% increase at 11mM Ten which is consistent with an increase in membrane microviscosity. The binding of Ten to PL also quenched the fluorescence intensity of DPH. These results support the concept that prothrombin activation occurs on a PL surface and suggest that membrane fluidity may affect the reaction rate.
Purification Of 1- And 0-Gla Prothrombins And Their Differentiation With Normal And Other Dicoumarol-Induced Prothrombins
Authors: Malhotra O P
Published: July 2018

In normal prothrombin, 10-glutamyl residues present in the amino portion of the molecule are carboxylated to form γ-carboxyglutamic acids (gla). Dicoumarol, an antagonist of vitamin K, induces the production of (partially) acarboxylated atypical prothrombins. In addition to our atypical varieties, viz. 7−,5− and 2-gla prothrombins, we have isolated two more atypical molecules, one containing 1.0±0.1 gla (1-gla prothrombin) and the other approximately 0.25 gla (0-gla prothrombin). These two variants adsorbed onto alumina Cγ-gel (Bio-Rad), similar to 2-gla protein, but were derived from 40 to 50% (NH4)2SO4 saturation. The purified materials, obtained after isoelectric precipitation followed by preparatory polyacrylamide-gel electrophoresis and heparin agarose chromatography, showed a single component by analytical disc-gel electrophoresis both in the presence or absence of sodium dodecyl sulfate (SDS) and contained antigenic activity comparable to that of normal prothrombins.

The pI’s of the two variants by column electrofocusing were each 4.835±0.015. Similarly, the two proteins did not reveal any difference in electrophoretic mobility; however, their prothrombin fragments 1 (F1, residues 1-156) did—0-gla F1 moved slower than 1-gla F1. Employing anti (normal) prothrombin sera with Ca2+ , the two, 0− and 1-gla, F1’s produced vaguely visible immunoprecipitates which were definitely lighter than all the other F1’s including 2-gla. These results confirm that not only are multiple forms of atypical prothrombin induced by dicoumarol but also that gla does affect the immunochemical properties of the gla-containing fragment.
Effect Of A Non-Activated Prothrombin Complex Concentrate (Prothrombinex) On Platelets: In Vitro And In Vivo Studies
Authors: Ekert H , Dean F L , Lane J L
Published: July 2018

Chromatography on Sephadex G-200 of the prothrombin complex concentrate, prothrombinex (Px) showed it to have inhibitor and potentiator fractions when tested by the non-activated partial thromboplastin time (NAPTT). The inhibitory effect was related to the heparin content of Px, as it was removed on ECTEOLA-cellulose. The potentiator fraction clotted fibrinogen and this could be inhibited by hirudin. Thrombin-like activity of this fraction was shown using chromogenic substrates. The effect of the potentiator fraction in the NAPTT was markedly enhanced by platelets. Diluted Px and the potentiator fractions caused aggregation of washed platelets which could be inhibited by hirudin. Aggregation was independent of the prostaglandin pathway, as it occurred in washed aspirin-treated platelets. Neither diluted Px nor potentiator fractions aggregated platelets in platelet rich plasma. Infusion of Px was followed by a rise in β-thromboglobulin (β-TG) in 2 of 3 patients two minutes after infusion. This rise could not be ascribed to the presence of β-TG in Px or to the heparin present in Px. These findings suggest that Px has a thrombin-like activity and that its mode of action in patients with factor VIII antibodies may result from its effect on platelets and their interaction with coagulation enzymes.
Inhibitor Of Factor VII Activation In Multiple Myeloma
Authors: McNutt R A , Penner J A
Published: July 2018

An investigation of a patient with IgG myeloma who developed a clotting abnormality is described. Factor VII activity was 4%. A prothrombin time of 20.4 seconds with control of 11.0 seconds was found in association with normal partial thromboplastin time, factor X level, and a failure to correct with VitaminK and fresh frozen plasma.

Methods for characterization were as follows. After defibrination and ammonium sulfate fractionation, plasma globulin portion was further purified on a DEAE ion exchanger. To identify inhibitor, Ig, alpha and beta globulins, myeloma proteins as well as albumin and fibrinogen isolated from patient plasma were incubated with normal plasma for two hours. Inhibitor activity was found in monoclonal antibody fraction. SPA affinity column removed inhibitor activity.

To elucidate whether inhibitor was directed against FVII, patient plasma, myeloma proteins, and monospecific xenogeneic antibodies to FVII were compared for their ability to bind 125I FVII. Free from bound forms were separated on a Sephacryl S200 column. Chromatograms indicated absence of binding of patient inhibitor to 125I tracer.

Normal and patient plasma were incubated with cephalo- plastin for 2 hours at 37°C. Prolonged recalcification time from 50 to 170 seconds was seen with patient plasma. More significantly, when inhibitor plasma and cephaloplastin were incubated for 2 hours and added to FVII deficient plasma, correction of prothrombin time was evident. Patient plasma not incubated with cephaloplastin failed to correct FVII deficient plasma.

We conclude that low activity obtained in the assay system was due to the myeloma protein which inhibited FVII/ thromboplastin interaction rather than an acquired inhibitor directly affecting FVII. It seems likely that the antibody is directed to the functional site of thromboplastin responsible for FVII activation.