bluuebunny/crossref_metadata_2025 huggingface dataset:
INCREASED THROMBIN GENERATION DURING COLLECTION OF BLOOD FROM DONORS TAKING ORAL CONTRACEPTIVES (OC)
Authors: Skjønsberg O H , Kierulf P , Engebretsen L F , Gjønnes G , Godal H C
Published: June 2019

450 ml blood was drawn into Fenwal PVC-bags from 26 OC-users and 28 non-users. The groups were comparable with regard to sex, age, smoking habits and blood collecting time. Thrombin generation was estimated as the fibrinopeptide A (FPA) concentrations in the bags immediately after ending the donations. The blood was also analyzed following storage at 4°C for 24 hours.

Subsequent to donation, the median FPA level in the control group was 4.2 (range 1.8-18.9) and in the OC-group 7.5 (range 2.2-113.7) (p°0.05), reflecting a more pronounced thrombin generation during collection of blood from OC-users. The level of prekallikrein (PKK) was also higher in blood drawn from OC-users (97.0% (73-128) in the control group versus 121.5% (79166) in the OC-group (p°0.001)), as was the level of FVIII:C, the latter difference was, however, not significant (p=0.06). The A T-111 concentrations were similar in the two groups.

No cold promoted activation could be observed following storage of the bags at 4°C for 24 hours, neither was any change observed in the levels of FPA, PKK or AT-III. There was no difference between the groups with regard to decay of FV111: C upon storage.

We conclude that thrombin generation is more pronounced during collection of blood from donors taking OC. Only some of the OC-users seem to generate appreciable amounts of FPA (19% above 30 ng/ml), and it is important to notice that all bags containing alarmingly high levels of FPA were drawn from women taking OC.
SERUM BINDING ABILITY OF PGI2 IS DECREASED IN NORMAL AND ABNORMAL STATES OF PREGNANCY
Authors: Saba H I , O’Brien W F , Knuppel R A
Published: June 2019

Several abnormalities of hemostasis have been observed in pregnancy. We have recently reported the findings of circulating platelet aggregates, thrombocytopenia and platelet exhaustion in pregnant states indicatedplatelet hyper-reactivity (Am J Ob Gyn 155:486;1986). Currently, we haveattempted to delineate the pathogenesis of this phenomenon. Sera from 36normal pregnant subjects and nine pregnant subjects with pre-eclampsia were examined for their ability to bind PGI2. Thirteen age-matched nonpregnant subjects' sera was used as controls. 350 pi of sera from these subjects was incubated for three minutes with 35 pi of radiolabeled sodium salt of PGI2. The mixture was then eluted on a Sephadex G25 column. Thirty samples of 0.35 ml were collected. Two peaks of radioactivity were obtained. The first peak was protein-bound PGI2, and the second contained unbound PGI2 and its hydrolytic product, 6ketoPGFia. Percent binding wasdetermined by (count in first peak/total count eluted) x 100. The albumin (ALB) and total protein (TP) was also measured to evaluate their relationship with the binding. Results indicate that PGI2 binding ability of sera from normal pregnant and pre-eclampsia subjects is markedly impaired. Pregnant groups when compared to controls exhibit statistically significant difference. The ALB and TP failed to suggest a relationship with binding abnormality. Studies being performed on platelet aggregation indicate that impairment of PGI2 binding in the subject's sera accompanied the loss of antiaggregatory influence of this PGI2. These studies suggest that plateley hyperreactivity in normal and abnormal pregnancy may be related to impairment of PGI2 binding in their blood, and cause unstability and rapid hydrolysis of this important platelet antiaggregatory agent available from the vascular wall.
PROTEIN C AND PROTEIN S LEVELS IN TALL GIRLS TREATED WITH ETHINYL -OESTRADIOL
Authors: Huisveld I A , Greven E C G , Bouma B N
Published: June 2019

Increased levels of several coagulation factors and reduced levels of anti thrombin III (ATIII) have been observed in adults on oestrogen medication and in girls who were treated for excessive tallness with high doses of ethinyloestradiol (E2). In females using oral contraceptives an additional effect on the protein C system has been reported. In our study we evaluated the effect of e-thinyloestradiol treatment (0.1-0.3 mg, administered for at least 6 months) in 14 tall girls (age 14 + 2 yr and height 179 + 4 cm). 13 girls of comparable age (13 + 2 yr) and height 175 + 7 cm) served as controls. Protein C, protein S, C4-binding protein as well as Factor II, plasminogen and ATIII antigen levels were determined by rocket immunoelectrophoresis. ATI11 heparin cofactor ami do-lytic activity was assessed using S2222. Protein S antigen not in complex with C4b-binding protein (PSfree), was assayed in the supernatant plasma after selective precipitation of the protein S-C4b-binding protein complex with PEG 8000. Complete separation of free protein S and PS-C4b-bp complex was verified with crossed immunoelectrophoresis. Results were expressed as % of a normal pool. In the group of girls who were being treated with ethinyl oestradiol (E2-users) significantly higher levels (P<0.001) were observed for protein C (126 ± 29 vs 88 ± 10 in the controls), Factor II (109 ± 18 vs 81 ± 12 in the controls) and plasminogen (131 ± 19 vs 85 ± 9 in the controls). Protein S (58 ± 10 vs 97 ± 8 in the controls) and PSfree (54 ± 11 vs 88 ± 11 in the controls) were sig nificantly reduced (P<0.001) in the E2-users. Only a slight reduction was observed in ATIII antigen levels (95 ± 16 vs 104 ± 9 in the controls) and in the ATIII heparin cofactor activity (105 ± 14 vs 104 ± 9 in the controls). No difference between E2-users and controls was observed in the C4b-binding protein levels (111 ± 25 vs 114 ± 33).

Results indicate that treatment of tall girls with high doses of synthetic oestrogens is associated with in vitro signs of hypercoagulability quite comparable to those observed in adult females. However, in contrast to observations made in adults no thromboembolic complications have been reported in juvenile subjects on E2-medication implying additional (aqe-related) variables in the qe-nesis of thrombosis.
TOTAL AND FREE PROTEIN S IN NEPHROTIC SYNDROME
Authors: Gadelha-Parente T , Gouault-Heilmann M , Rostoker G , Levent M , Rafowicz S , Intrator L , Lagrue G
Published: June 2019

25 consecutive patients (15M, 10F ; mean age 30 years) with nephrotic syndrome (NS) of different grade were studied. Control group consisted in 18 healthy adult volunteers. Total protein S antigen (TPS:Ag) and free protein S antigen (FPS:Ag) after precipitation of C4-BP-bound protein S by PEG 3-5 % final concentration were measured by Laurell's technique. PS:Ag was also searched in concentrated urine of 9 patients by ELISA method, more sensitive than the Laurell's technique. In the same plasma samples we measured C4-BP, Protein C Ag and AT III biological activity (all reagents from D.Stago). Serum albumin level, proteinuria, proteinuria selectivity index, triglycerides, cholesterol levels were recorded. TPS:Ag was found elevated in NS (1.30±0.3 U/ml) in comparison with control group (1.09±0.32 U/ml) and the difference was statistically significant (p<0.05). The mean values of FPS:Ag observed in patients and controls were not statistically different, but if we consider 95 % confidence limits (0.99-1-35 U/ml), 16 pts had normal or elevated FPS:Ag level, whereas 9 had decreased FPS:Ag level. A positive correlation was found between TPS:Ag and FPS:Ag in control group (r=0.66 ; p< 0.001) and in patients with NS (r=0.4l, p<0.05). C4-BP was significantly (p<0.01) increased in nephrotic patients ( 1.37 ± 0.36 U/ml) in comparison with control group (1.04±0.27 U/ml). A negative correlation was found between FPS:Ag and C4-BP levels in control group (r = −0.57, P< 0.01) but not in nephrotic patients. A positive correlation was found between FPS:Ag and albumin level and between FPS:Ag and cholesterol level. No correlation was found between TPS:Ag or FPS:Ag and proteinuria, proteinuria selectivity index, AT III and protein C levels. Traces of PS were found in urine (0. to 2.5 U/day) in 9 patients tested. 2/25 pts suffered thromboembolic events : one had a very low level of FPS:Ag in addition to a decreased level of AT III. The other one had normal FPS:Ag and AT III level but a borderline Protein C level. In conclusion. An acquired FPS:Ag deficiency was observed in 9/25 pts with NS despite an increased level of TPS:Ag. In this small series of patients the acquired FPS deficiency does not seem to be related either to an urinary loss of FPS or to an increased binding to C4-BP, as suggested by some authors.
EVALUATION OF COMMERCIAL PROTEIN C ASSAYS
Authors: Morrien-Salomons M M , Sturk A , Huisman M V , Borm J , Büller H R , ten Cate J W
Published: June 2019

Plasma protein C inactivates the activated coagulation factors V and VIII. The assay of protein C is important, because a protein C deficiency is associated with a thrombotic tendency. We therefore evaluated 5 commercial assays in 49 normal volunteers, 48 patients suspected of deep vein thrombosis (DVT) of the leg but with negative impedance plethysmography (IPG), and 52 patients with DVT proven by IPG. The assays were rocket electrophoresis (Merz and Dade antibody), ELISA (Boehringer Mannheim), 2 chromogenic activity assays (Behringwerke and Kabi) and a clotting assay (Behringwerke). Coumarin therapy was used by 13 DVT positive, and 3 DVT negative patients. Results are presented in the table.

TABLE: In correlation 1 and 2, the assay results all non-coumarin treated individuals (n = 133) were compared with rocket electrophoresis and ELISA resp.

In the non-coumarin treated patients, both in the DVT positive and in the DVT negative patient group one protein C deficiency was detected by all assays.

Based upon the large assay VC (ROCKET) and normal range (B. CLOT), and poor correlation of the assays with the ELISA (ROCKET, B. CLOT) we conclude that the ELISA, B.CHROM and K.CHROM are to be preferred. However, as B.CHROM does not need a plasma absorption step it is somewhat preferable for activity assays.
IDENTIFICATION OF VITAMIN K-DEPENDENT CARBOXYLASE ACTIVITY AND EVIDENCE FOR PROTHROMBIN SYNTHESIS IN ALVEOLAR TYPE II CELLS
Authors: Wallin R , Rannels S R
Published: June 2019

Prothrombin precursors have been shown to be present in micro-somes from lung alveolar type II cells. Immunoblotting revealed two microsomal precursors of apparent mol. wt. 68 and 65 kDa. The 65 kDa protein appears to be the substrate for the vitamin Independent carboxylase. Fluorography of [14c]-labeled precursors of vitamin K-dependent proteins in lung microsomes shows that the lung has several precursors that are not found in the liver. Pulmonary macrophages appear to be devoid of vitamin K-dependent carboxylase activity. However, type II epithelial cells have significant activity and this activity was unaltered when these cells were maintained in primary culture for 3 days, suggesting that carboxylase activity is expressed in alveolar epithelium independent of culture-induced changes in cellular differentiation. Carboxylase activity in type II cells was enhanced 2-fold when cells were cultured for 24 hours in the presence of 50 μM warfarin. Type II cells, therefore, resemble hepatocytes with regard to their response to coumarin drugs. Our data suggest that macrophages and type II cells may act in a cooperative manner to contribute to extravascular coagulation in the lung. Supported by NIH Grant HL-32070-02 and by The American Heart Association.
A New Type of Congenital Dysfibrinogenemia (Fibrinogen Tokyo) with Defective Stabilization of Fibrin Polymers
Authors: Samori T. , Yatabe M. , Ukita M. , Fujimaki M. , Fukutake K.
Published: June 2019

A new type of congenital dysfibrinogenemia characterized by abnormal stabilization of fibrin polymers under the normal concentration of plasma factor XIII and normal thrombin time has been discovered in Tokyo.

The molecular abnormality of this abnormal fibrinogen are shown by an anodal immunoelectrophoretic mobility and an abnormal pattern of D fragment in fibrinogen degradation products by plasmin digest on Immunoelectrophoresis and crossimmunoelectrophoresis.

However, the plasma fibrinogen level of this case measures always in the normal range, when immunological methods or thrombin dependent methods are used, and the decreased level of plasma factor XIII is indicated by the use of assay methods based on clotlysis.
The Variability of Platelet-Collagen Interaction. Its Influence on Aspirin-Induced Inhibition of Platelet Aggregation
Authors: Sack E. S. , Quiroga E.
Published: June 2019

A study of platelet aggregation induced by progressive concentrations of collagen was performed in 40 normal persons equally grouped by sex immediately before and 90 min after the ingestion of 0.500 g aspirin. Simultanously serum ASA-esterase activity and plasma salicylic acid levels were investigated in the pre- and post-ASA samples respectively. Wide individual variations in platelet responsiveness to collagen were found when the minimal amount of the aggregating agent necessary to provoke significant aggregation was located. The percentage of aggregation and the slope were the most sensible indicators of both the individual variances and the ASA-induced inhibition. Correlation was found in both groups between the minimal amounts of collagen required for initiating aggregation pre-and post-ASA (men: r = 0.73, p < 0.01; women:r = 0.55, p < 0.05). Also a positive correlation was found between the minimal collagen concentration required to initiate aggregation pre-ASA and the aspirin-induced inhibition in the aggregation produced by a higher collagen concentration (men: r = 0.63, p < 0.01; women: r = 0.75, p < 0.01). Important individual and sexual differences were detected in the serum content of ASA-esterase activity. Men had 114.05±3.14 μg/salicylic acid released/ml/60 min whereas women 88.65±4.93, p < 0.01. A negative significant correlation was demonstrated between the ASA-esterase activity and the differences in collagen concentrations necessary to start aggregation pre- and post-aspirin in the men group (men: r = −0.67, p < 0.01; women: r – −0.39, p > 0.05). Failure to consider individual variances in platelet reactivity may lead to false conclusions about the ASA effectiveness as an anti-thrombotic agent.
Role of Platelets in Recurrent Deep Vein Thrombosis
Authors: Wu K. K. , Barnes R. W. , Hoak J. C.
Published: June 2019

To evaluate the role that platelets play in the pathogenesis of recurrent deep vein thrombosis (DVT), a platelet count ratio method was used for the detection of platelet aggregates and an aggregometric technique was used to measure spontaneous aggregation (SPA) in 27 patients with idiopathic recurrent DVT. Seventeen patients were found to have decreased platelet aggregate ratios (mean 0.63±SEM 0.02) which were significantly lower than those of normals (0.90 ±0.02, p < 0.01). Twelve of the 17 patients had SPA. The mean platelet survival half-time of 5 patients with increased platelet aggregates was 2.9 days±0.49, significantly decreased from that of normals (4.2 ±0.10, p < 0.05). Platelet survival values were normal in patients with normal platelet aggregate ratios. Five patients who failed to improve on oral anticoagulant therapy responded to aspirin and dipyridamole with normalization of platelet aggregates and disappearance of SPA. An additional patient responded to sulfinpyrazone. When the drug was discontinued, pulmonary embolus recurred. These findings suggest that recurrent DVT may involve heterogeneous groups of patients and platelets may play an important pathogenetic role in some of them. The approach to the problem with this panel of 3 tests appears useful in the selection of patients for treatment with antiplatelet agents.
A Series of Extremely Potent Thrombin-Inhibitors Newly Synthetized
Authors: Okamoto S. , Hijikata A. , Kikumoto R. , Tonomura S. , Tamao Y.
Published: June 2019

Starting with the simple arginine derivatives having weak thrombin-inhibitory activity, ca. 320 chemical compounds were designed and synthetized by the authors, in such a way as increasing the inhibitory activity by stepwise chemical modifications. The extremely potent inhibitors thus obtained are described.

These inhibitors are chemcially naphthalene-sulfonyl-arginine derivatives, the basic structure being composed of three functional sub-units and designed, to reflect the part of fibrinogen peptide to be split by thrombin.

One of the representatives, dansyl-arginine-ethyl-piperidine amide, inhibited 50% of thrombin activity at 0.1 μM when 5 μM fibrinogen was substrate. Similar result was also obtained when benzoyl-Phe-Val-Arg-pNA was substrate. The mode of inhibition was found competitive.

The inhibitors suppressed the platelets aggregation due to thrombin satisfactorily. However, the inhibition on plasmin, reptilase or trypsin was far weaker, indicating the very high selectivity to thrombin. Their relatively low toxicity led the authors to extend the studies toward the animal experiments and results obtained will be reported elsewhere.
On the Plasmin Digestion of Factor XIII
Authors: Mikami K. , Mikami T. , Suzuki H. , Fujimaki M. , Fukutake K.
Published: June 2019

The plasmin digestion of factor XIII in the plasma with congenital afibrinogenemia has been reported by Suzuki et al. in 1967. The present investigation using polyacrylamide gel electrophoresis and crossimmunoelectrophoresis with anti-factor XIII (A & S) serum demonstrates that the process of plasmin digestion of factor XIII can be divided into three steps ; the subunit A of factor XIII such as placental or platelet factor XIII is ready to get plasmin digestion following the decrease of amount and activity of subunit A, and the subunit S in the A1
S complex like plasma factor XIII is more readily affectable on the plasmin digestion than the subunit A of the complex, and the A2
S complex coexisting with fibrinogen or plasma protein is fairly stable on exposure to plasmin.
Common Action of Inducers and Inhibitors of Aggregation on Phosphorylation of Surface Localized Ca-Binding Protein
Authors: Booyse F. M. , Marr J. , Tomlinson D. , Yang D. G. , Rafelson M. E.
Published: June 2019

Analysis of intact 32P-labeled platelets by SDS-polyacrylamidegel electrophoresis shows the presence of only a single phosphorylated protein. This phosphoprotein has been isolated, has a subunit molecular weight of 11,100, is surface localized (lactoperoxidase-125 Iodine labeling) and binds 1 mole of Ca2+ per mole of phosphorylated subunit. The γ-32ऩ from (γ-32P) ATP is specifically incorporated as 0-phosphoserine by a cAMP-dependent protein kinase.

The phosphorylated state and hence the Ca-binding of this protein is maintained by membrane associated cAMP-dependent protein kinase phosphorylation and phosphoprotein phosphatase dephosphorylation. Thrombin, collagen, histone and ADP competitively inhibit phosphorylation and Ca-binding by serving as protein kinase acceptors. Epinephrine, serotonin and ristosetin induce dephosphorylation and abolish Ca-binding by activating the phosphoprotein phosphatase. PGEl5 ATP, cAMP increase phosphorylation and Ca-binding by activating the protein kinase ; aspirin and EDTA increase phosphorylation and Ca-binding by inhibiting the phosphoprotein phosphatase. In 3 cases of Glanzmann’s thrombasthenia, increased phosphorylation and Ca-binding was due to decreased phosphoprotein phosphatase activity-activity was restored the addition of ristosetin. A common phosphorylation-dephosphorylation mechanism for the action of inducers and inhibitors on Ca-mobilization and platelet activation will be presented.
Platelet Cytogel Extrusion : Fact or Artifact ?
Authors: Booyse F. M. , Sedlak B. , Tomlinson D. , Rafelson M.E.
Published: June 2019

Analysis of human platelets treated with thrombin (0.25 U/ml) for 4-12 seconds (in the presence of ATP or EDTA), fixed with formaldehyde and analyzed on silicone gradients indicates the rapid appearance of teinaceous material outside the platelets which contains immunochemical identifiable thrombosthenin and lactic acid dehydrogenase. Replicas of whole mount shadow-casted platelets, treated with thrombin for 8-10 seconds in the presence of 1 × 10-5 M ATP and 5 × 10-5 M Mg2+ show small aggregates (2-4 platelets) entrapped in what appears to be a gel consisting of a meshwork of 400-600 Å filaments indistinguishable from microfilaments obtained when platelet cytoplasm or purified thrombosthenin is treated in a similar manner. Platelets, preincubated with horseradish peroxidase (to label only outer surface of cell), were treated with thrombin, fixed (gluteraldehyde) 4-12 seconds after the addition of thrombin. The thrombin activation sequence was followed by immunohistochemical staining using monospecific antihuman platelet myosin serum (rabbit) and ferritin-conjugated antirabbit gamma globulin serum (goat). Preliminary observations under the electron microscopie show the rapid appearance (4-8 seconds) of platelet myosin beyond the intact cell membrane (limited by horseradish peroxidase staining). This myosin containing material (cytogel ?) appears to interact with other similarly activated platelets. We have concluded from these observations that platelets extrude a portion of their contents beyond their plasma membranes and that this extruded material interacts to form the intercellular bonds between aggregating platelets.
Cleavage of Thrombosthenin A and A’ by Thrombin
Authors: Muszbek L. , Fésüs L. , Ölveti E. , Szabó T.
Published: June 2019

Bovine platelet actin prepared by Spudich’s method is separated into two peaks on a Sephadex G-200 column. The actin of both peaks had a m. w. of 42000 on SDS PAG and activated myosin ATP-ase although in a quantitatively different manner. Actin eluated in the first peak (A’ - probably in oligomeric form) was not polymerized in 2 mM MgCl2 and 0.05 M KCl while that of the second peak (A) went through normal G-F transformation. If CaATP was present in the incubation mixture neither actin was attacked by thrombin. In the presence of EDTA G-actins became attractive for thrombin, “normal” F-actin was not split and A’ was cleaved in 0.1 M KCl, too. The pattern of cleavage of both A and A’ was the same as it was found for muscle actin in our earlier study (Proc. Natl. Acad. Sci. 71, 2208 1974), and the final split-products were: actinopeptide I, II and two fragments of 26500 and 11000 m. w. It is suggested that the possible attractivity of membrane associated platelet actin for thrombin may have an importance in thrombin induced platelet aggregation.
Venous Thrombo-Embolism in Childhood
Authors: Macintyre I. M. G. , Jones D. R. B. , Ruckley G. V.
Published: June 2019

Venous thrombo-embolism has been considered to be rare in infancy and childhood. Hospital in-patient statistics in Scotland over a 4-year period were examined. Forty-nine patients aged 15 years or less had been coded as venous thrombosis. After computer and clinical errors had been removed 36 cases remained. Renal vein thrombosis accounted for 12 of these, caval thrombosis following ventriculo-atrial shunt 4, cerebral thrombophlebitis 3, umbilical vein thrombosis 2, pulmonary thrombosis in infancy 2, axillary vein thrombosis 2 and jugular venous thrombosis 1. There were 10 cases of deep vein thrombosis of the lower limb and a clinical study of these is the subject of this paper. Two patients also had pulmonary embolism and two others developed chronic venous insufficiency. Children at high risk are those with sepsis or trauma. Venous thrombosis may simulate osteomyelitis and pulmonary embolism may be misdiagnosed as bronchopneumonia. Clinicians must be aware of the possibility of thrombo-embolism in childhood if correct diagnosis and treatment is to be instituted.
Failure of Aspirin and Dipyridamole to Influence the Incidence of Leg Scan Detected Venous Thrombosis after Elective Hip Surgery
Authors: McBride J. A. , Turpie A.G.G. , Kraus V. , Hiltz C.
Published: June 2019

Drugs known to affect platelet function have been shown to be effective in the prevention of venous thrombosis after surgery. A double-blind randomized trial of aspirin plus dipyridamole was carried out to investigate the prophylactic value of the drug combination in the prevention of venous thrombosis detected by 125 Iodine-labelled fibrinogen leg-scanning after elective hip surgery. Aspirin 900 mg plus dipyridamole 100 mg were given once pre-operatively and twice daily for 10 days postoperatively, or until the patient was fully ambulant. 43 patients were entered into the trial, 22 in the control group and 21 in the treated group. Both groups were similar with respect to age, sex and factors known to predispose towards venous thrombosis. 8 patients (36.7%) in the control group and 8 patients (38.1% ) in the treated group developed leg scan-detected deep vein thrombosis. The thrombi were detected between the 1st and 17th post-operative day and were found as frequently in the operated limbs as in the non-operated limbs in both groups. It is concluded that prophylaxis with aspirin 900 mg and dipyridamole 100 mg twice daily does not influence the incidence of leg scan-detected deep vein thrombosis after elective hip surgery.
A New Fibrinogen Anomaly: Fibrinogen Buenos Aires
Authors: Buraschi J. A. , Sack E. S. , Quiroga E. , Hendler H.
Published: June 2019

Report of a familial defect of fibrinogen in a 42 year old woman and in one of her siblings. The defect in the boy produced different abnormalities. She had a lifelong hemorrhagic tendency and deffective wound healing and only slight prolongation of the thrombin time. She had normal fibrinogen levels (tested by several methods) and normal coagulation factors (including Factor XIII). Both fibrinolytic mechanism and platelet function were normal. Plasmatic antithrombin III activity was normal whereas its serum activity was increased. The bleeding time was moderately prolonged and clot retraction impaired. The thromboelastogram showed reduction of the “ma.” and plasma polymerization was deficient. These last two abnormalities were partially corrected by transfusions of fresh plasma. Aggregation of fibrin monomers was also deficient whereas fibrinopeptide release was quantitatively normal. The abnormal fibrinogen has increased immunoelectrophoretic mobility and an abnormal sedimentation pattern.

Her son had a markedly prolonged thrombin time with bovine thrombin whereas with the human enzyme the test was moderately abnormal. Plasma polymerization was absent and sedimentation cooefficient increased. Reptilase time was normal. The abnormal fibrinogen is tentatively designated fibrinogen Buenos Aires, since its possible identity with other abnormal fibrinogen has not been excluded.
Electron Microscopic Studies of Fibrinogen Degradation Products
Authors: Marder V. J. , Budzynski A. Z. , Tranqui-Pouit L. , Suscillon M. , Hudry-Clergeon G.
Published: June 2019

Many of the details regarding the organization and tertiary structure of fibrinogen are still controversial. Regarding the disulfide bonds which link the two halves of fibrinogen, one group considers that such bonds link the amino terminal parts together and that plasmic degradation leads to the formation of two Fragment D derivatives. The alternate model considers the two halves of fibrinogen to be linked by disulfide bonds in two regions and that plasmin liberates a single dimeric Fragment D molecule. This question was approached by a study of the electron microscopic appearance of fibrinogen and its plasmic derivatives. Fragment X obtained from Stage 1 digests and fibrinogen were both globular, while Fragment X of Stage 2 digests appeared as a nodular filament. The Stage 1 and Stage 2 Fragment X preparations had approximately the same molecular weight, but could be differentiated by subtle differences in polypeptide chain structure. Fragments Y and D were also filamentous, although shorter than Fragment X (Stage 2), and Fragment E appeared as a small clump of nodules. These results agree with the concept that fibrinogen consists of a strand of nodules connected by thin filaments, folded into a compact, spherical shape. The molecule opens up when stabilizing bonds are disrupted or liberated by plasmin. The data are compatible with a fibrinogen molecule in which the two halves are linked by a single locus of disulfide bonds at the amino terminus and with the asymmetric hypothesis of plasmic degradation to Fragments X, Y, D and E.
Comparative Investigations on Time Dependant Changes of Enhanced Aggregation
Authors: Scharrer I. , Breddin K. , Ziemen M.
Published: June 2019

Comparing spontaneous platelet aggregation measured in PAT III (Breddin, 1975) and ADP-collagen- and epinephrine induced aggregation a close correlation was found if the time dependent changes of the four tests were considered. The percentage of desaggregation in ADP-induced aggregation (10-6 M) correlated well with the extent of spontaneous aggregation if recorded 30 minutes after blood sampling. The time lag between addition of collagen lug/ml correlated best with spontaneous aggregation if measured 30 minutes after blood sampling. In epinephrine-induced aggregation (5 × 10-6 M) the angle of second wave of aggregation is well correlated with the results of PAT III 60 min after blood sampling. In risk factor screening for thromboembolic diseases reliable results can only be obtained if the aggregation tests are performed within certain time limits.
Activation of Dicoumarol-Induced Atypical Prothrombin
Authors: Malhotra O. P.
Published: June 2019

One of the dicoumarol-induced atypical prothrombins isolated from bovine plasma did not bind Ca++ even when the Ca++ concentration in medium was 13.5 mM. The normal prothrombin under the same conditions bound 9 nmol/nmol protein. This atypical material, similar to other variants, activated slowly in the presence of thromboplastin, Ca++, factors V, VII and X. It contained 85% antigenic activity but only 16% bioactivity, lower than other variants which bind some Ca++. The bioactivities correlated with the Ca++ binding capacities, while the generation of thrombin by Echis carinatus venom etc. depended upon the immunological activities of the purified proteins. On the other hand, the semi-purified preparations of the atypical prothrombin showing two or more components by disc-gel electrophoresis, and containing 60% antigenic activity, did not activate by biological means. In the presence of thrombin, both the normal and the atypical variant yielded Intermediate I and Fragment I. When factor Xa was added, Intermediate I produced Fragment II and Intermediate II. The latter subsequently is transformed into thrombin. These and other studies show that the dicoumarol-induced prothrombins should have some biological activity, and this activity depends upon the extent of defect in their Ca++ binding abilities.

(Supported by V. A. Project 5445-01.)
The Phlebo-Dynastat: A New and Effective Method of Prophylaxis for Deep Vein Thrombosis
Authors: McKenna R. , Bachmann F. , Kaushal S. P. , Galante J.
Published: June 2019

Peripheral deep vein thrombosis (DVT) is a frequent complication of orthopedic surgery. Evaluation of new methods of prophylaxis is necessitated by the disadvantages of the popular low dose heparin method.

In a previous study we found a 50% (20/40) incidence of DVT in patients undergoing total hip or knee replacements. In this high risk group, we undertook a prospective study of the prophylactic value of the Phlebo-Dynastat - a previously undescribed intermittent pneumatic compression device which alternately inflates thigh and calf cuffs to a maximum pressure of 30 mmHg. Seven patients had total knee replacements and 19 had total hip replacements. 125I-fibrinogen scanning was started pre-operatively in all patients. Venography was performed on 25 limbs in 19 patients. Patients were assigned to treated or untreated groups by random numbers. Seven of 12 untreated patients developed DVT complicated by pulmonary embolism in 3, but only 1/14 patients in the treated group developed DVT. This difference is significant at the 0.009 level (Fisher’s exact probability). DVT did not occur in 3 treated patients with shortened activated partial thromboplastin time but occurred in 2/3 untreated patients.

We are reporting a new previously undescribed method for preventing DVT and have demonstrated its effectiveness.
Efficacy and Monitoring of Heparin Therapy in Experimental Venous Thrombosis
Authors: Chiu H. M. , Yung W. , Hirsh J.
Published: June 2019

Opinions differ regarding the most effective means of administering heparin therapy. We have compared 1) the effectiveness of continuous intravenous infusion with intermittent intravenous injection and 2) the relative effectiveness of using heparin level and activated partial thromboplastin time (APTT) for monitoring heparin in experimental venous thrombi in rabbits. Thrombus extension was quantitated by measuring the net accretion of 125I rabbit fibrinogen onto non-radioactive experimental venous thrombi over a 10 hour period. Fifty-four animals received heparin and 30 controls received saline. Heparin was administered by 1) continuous infusion in the following doses: a) initial bolus 200 U/kg followed by 400 U/kg infused in 10 hrs (full dose), b) 3/4 of full dose and c) ½ of fulldose; 2) by intermittent intravenous injection of a) 200 U/kg, b) 150 U/kg and c) 100 U/kg 4 hourly. All 3 continuous intravenous regimens showed significantly less accretion than the control animals, however, of the intermittent regimens only the full dose regimen showed significantly less accretion than the controls. Continuous heparin in both full dose and 3/4 dose showed significantly less accretion than the corresponding intermittent intravenous heparin regimens. The antithrombotic effect of heparin was reflected by both the heparin level and the APTT. The experiments were repeated with continuous intravenous heparin following infusion of cryoprecipitate into rabbits. This shortened the pre-heparin APTT and reduced the post-heparin APTT rise. Optimal suppression of thrombus extension in both normal and cryoprecipitate treated rabbits was obtained when APTT was maintained at approximately twice pre-treatment level.
Resistance to the Development of Spontaneous Atherosclerosis in Pigs with von Willebrand’s Disease
Authors: Bowie E. J. W. , Fusier V. , Owen C. A. , Brown A. L.
Published: June 2019

The thoracic and abdominal aortas of 26 pigs with von Willebrand’s disease were examined for atherosclerosis by gross inspection. In 6 of these pigs, older than one year, a detailed histological study was made from tissue taken from areas most likely to develop atherosclerosis in the pig i.e. the distal part of the lesser curvature of the aortic arch and the posterior descending thoracic aorta at the level of the fifth intercostal artery. Six normal pigs were matched with the 6 von Willebrand pigs by breed, age, sex, and heart weight and a similar gross and histologic study of their aortas was performed. The aortas of 5 of the 6 normal pigs showed atherosclerotic lesions consistent with the known high frequency of atherosclerosis in normal pigs about one year of age. Only one of the bleeder pigs showed an atherosclerotic lesion; this was a fatty streak at the origin of the innominate artery. These preliminary observations suggest that pigs with von Willebrand’s disease are more resistant than normal pigs to the development of atherosclerosis. This may be related to the impaired platelet function in von Willebrand’s disease. We are extending these observations by means of prospective controlled dietary studies.
Clinical Trial in a Patient with Reduced Osmotic Resistency of Platelets
Authors: Sutor A. H. , Altemeyer K. H. , Böttcher D.
Published: June 2019

5 from 8 children of a family presented with severe bleeding symptoms, which repeatedly required blood transfusions. In all members of the family the following tests were normal: Plasmatic coagulation factors, several platelet function tests (spontaneous and ADP-aggregation, clotretraction, spreading) and capillary resistency. All children with bleeding tendency had a prolonged bleeding time (over 15 min) and a reduced osmotic resistency of platelets, in addition the hemorrhagometry (method of Sutor et al.) showed a prolonged bleeding time with increased bleeding intensity from a standardized small skin wound, which remained at the same high level until the test was interrupted after 60 minutes.

In one child, who had bled profoundly from the nose for hours, we gave freshly prepared cryoprecipitate. Before administration of the cryoprecipitate, hemorrhagometry showed a constant high blood flow for 30 minutes. 8 minutes after cryoprecipitate had been given the bleeding intensity decreased, after 5½ more minutes hemostasis was complete. At the same time bleeding from the nose stopped. Bleeding time now was within normal limits (5 min). The corrective effect of cryoprecipitate could repeatedly be demonstrated, its duration, however, was rather short: 6 hours after the transfusion the bleeding time became abnormal again.
The Effect of Hypoglycaemia on Platelet Count, Platelet Adhesiveness and Bleeding Time
Authors: Arapakis G. , Andriopoulos J. , Papayannis A. G. , Gardikas G.
Published: June 2019

The effect of hypoglycaemia (HG) on platelet count (PC), platelet adhesiveness (PAd) (Salzman’s method) and bleeding time (Ivy’s method) was studied in 30 healthy volunteers (12 males and 18 females), aged 17 to 55 years. Insulin cryst. 0.2 u/Kg b.w. was given i. v. and the platelet tests were performed immediately before insulin administration and during maximum HG. In 7 subjects PC and PAd were repeated immediately after the HG was reverted and 2h and 24 h later. It was found that PC and PAd were significantly increased during HG (p < 0.001); especially PC increased from m = 251 ±95X103 to m = 288±95X103, and PAd from m = 48±13% to m = 72±11%. These increased values persisted for 2h after HG was reverted by glucose infusion and returned to preinsulin levels 24 h later. On the other hand the bleeding time showed a significant shortening from m = 3.2±0.9 min to m = 2.1 ±0.8 min during HG {p < 0.001). There was no correlation between the changes of the various parameters studied.
Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin
Authors: Williams L. , Murano G.
Published: June 2019

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.

(Supported by USPHS N. I. H. Grant HL 14142.)
Variants of von Willebrand’s Disease in Sweden
Authors: Holmberg L.
Published: June 2019

In a recent Swedish investigation we could assign 39 families with all together 100 affected members to the elassical type of von Willebrand’s disease with low f VIII antigen ( < 50% ), low f VIII activity, prolonged BT, decreased platelet retention and ristocetin aggregation and typical response to infusion. A second type was found in 4 families (8 affected members). F VIII antigen was in the normal range and f VIII activity was slightly low or low normal. The other diagnostic criteria were present. A third variant was characterized by low f VIII antigen, prolonged BT and reduced platelet retention but normal f VIII activity and only slightly reduced ristocetin aggregation. The disease was symptomatic only in homozygous form. A fourth variant characterized by low f VIII activity, prolonged BT, normal f VIII antigen and normal ristocetin aggregation, was found in 7 families. As some of the pedigrees strongly suggests X-chromosomal inheritance, a haemophilia variant should rather be considered. In addition to the Swedish material some descendants of von Willebrand’s original families on Aland (Finland) have been examined.
In Vitro Formation of Fibrin-Monomer Complexes in the Presence of Isotope Labelled Fibrinogen-Fibrin Degradation Products. An Agarose Gel Filtration Study
Authors: Asbeck F. , Loo van de J.
Published: June 2019

Human citrated plasmas were mixed with purified 131I-fibrinogen and 131I-fibrinogen degradation products (FDP) or 125I-fibrin degradation products (fdp). After incubation with small amounts of thrombin (0.01–0.02 imits/ml Pl.), these mixtures were gel filtrated on Biogel A5m columns and the elution patterns of the 131I- and -labelled materials were determined.

In control experiments without thrombin incubation, no complex formation between fibrinogen, FDP or fdp in citrated plasmas could be detected. This was even true for fdp with a higher molecular weight than fibrinogen.

After thrombin incubation, up to 11% fibrin-monomer complexes were formed. Irrespective of their molecular weights, labelled fdp were not incorporated into these complexes.

Only large FDP – presumably derivative X – did partially copolymerize with fibrin-monomer complexes in citrated plasmas.
The Role of Calcium on the Release of Tissue Factor (Thromboplastie) Activity from Leukocytes
Authors: Muhlfelder T. W. , Khan I. , Niemetz J.
Published: June 2019

It is now well established that leukocytes may generate tissue factor (thromboplastic) activity (TFa), particularly after “stimulation” with endotoxin. As these leukocytes have also a thrombogenic effect, it appeared desirable to study the conditions in which the TFa was released. Leukocytes were obtained from the peritoneal cavity of rabbits after 2 doses of endotoxin and prepared as described previously (Nature New Biol. 232, 247, 1971). The leukocytes were incubated in siotonic buffered solutions, for up to 30 min then sedimented by centrifugation and the supemate removed for testing. The TFa was tested by the one stage test and the 2 stage assay. Significant release of procoagulant activity occured only when the leukocytes were suspended in solutions containing calcium. 2 mM of calcium caused maximum release of TFa while lower or higher concentrations were less effective. Other bivalent cations (Mg, Mn Zn) could not replace Ca ions and caused no release, while Co caused only very slight release of TFa. The release was time and temperature dependent: maximum activity being released at 37°C and almost no activity released at 4° C. When the effect of pH was studied, maximum release of TFa occurcd at pH 6.5–7.5. Substances which affect cell contractility such as vincristine, colchicine, cytochalasin B, as well as ouabain which affects some ATPase’s had no effect on release. These data show tlrat leukocyte tissue factor activity can be released from the cell in the presence of calcium. This release mechanism may play a role in some pathological conditions.

(Supported in part by NIH Grant 13155 and VA 6060–01.)
The Action of Brinase in Vitro and in Vivo
Authors: Gaffney P. J. , Lord K. , Thornes R. D.
Published: June 2019

Brinase (an extract of Aspergillus Oryzae) was shown to rapidly digest human fibrinogen in vitro to aggregable degradation products with a molecular size range of 310,000 to 230,000 the latter fragments being more slowly digested to core fragments, Dbr and Ebr. The fibrinogen polypeptide chain susceptibility to Brinase attack was in the order Aα, γ, Bβ, Lysis of the Bβ chain seems to be the rate limiting step in the conversion of the high molecular weight fragments (MW 310,000–230,000) to the core fragments Dbr and Ebr. The conservation of NH2 terminal Tyrosine during fibrinogen digestion and the very transient existence of D dimer fragments during totally crosslinked fibrin lysis suggest that the carboxy end of the γ chain is prone to Brinase attack. The crosslinked α chains of fibrin, while resistant to plasmin, are vigorously digested by Brinase. The plasma of cancer patients being treated with Brinase contained degraded fibrinogen (lacking intact Aα chains) and their aggregates. These aggregates contained some crosslinked γ chains (γ-γ dimers) suggesting that Brinase in vivo exorcises both a lytic and coagulant effect. Thrombin mediated clots in all the plasmas examined contained no crosslinked α chains. Positive plasma ethanol gelation tests can be explained by the presence of the aggregable high molecular weight fragments observed during the in vitro lysis of fibrinogen by Brinase.
Relative Affinity of Plasmin for the α1-Macroglobulin, Cl Inactivator and α1-Antitrypsin Inhibitors
Authors: Harpel Peter C.
Published: June 2019

The quantitative contribution of three major plasma protease inhibitors in binding plasmin has been studied. Mixtures of plasmin and each of the purified inhibitors were analyzed by SDS-acrylamide gel electrophoresis. Plasmin remained bound to its inhibitors in the presence of SDS and urea. A 1 : 1 molar ratio for complex formation was established, and treatment of the complexes with a disulfide bond reducing agent showed that the light chain of plasmin contained the binding sites for both CĪ inactivator and α-antitrysin. Limited degradation of all three inhibitors by plasmin was observed, and the altered inhibitor remained complexed to the enzyme. The competitive binding of 125I plasmin to mixtures of these inhibitors was followed by sucrose density ultracentrifugation and by SDS-gel electrophoresis. In mixtures containing physiologic molar ratios of enzyme and inhibitors, over 80% of the bound plasmin was complexed to the α2-macroglobufui (α2M). No evidence for an exchange of plasmin between the inhibitors was obtained.
Cold Promoted Activation of Factor VII and CĪ Esterase Inhibitor
Authors: Royen van E. A. , Leeuwen Kempes-van W. , Vos M. , Pondman K. W.
Published: June 2019

The cold promoted activation (CPA) of factor VII involves the conversion of prekallikrein into kallikrein by factor XII (Gjønnaess et al.). Since CI esterase inhibitor (CĪINH) and macroglobulin block the prekallikrein activating activity of factor XII, the role of CĪ2INH in CPA was investigated especially because CĪINH deficient plasma was available. Purified CĪINH was an effective inhibitor of CPA in final concentrations of 10–100 U/ml, only if added before the incubation of CPA positive plasma at 0° C for 20 hours. Addition of CĪINH after CPA generation did not affect the shortened Thrombotest Time (TT). Synthetic amidino compounds (diOHstilbamidine, dibromopropamidine), which have been claimed as specific inhibitors of CĪ esterase, were also effective inhibitors of CPA in final concentrations of 10-410-5 M. Alike to CĪINH they had no effect on a TT shortened by previous generation of CPA.

In 8 subjects with CĪINH deficiency the shortening of the TT of plasma incubated at 0°C for 20 hours exceeded that ina control group of 48 men and women not using oestrogen ic drugs (p 0.05). CĪINH activity proved to be consumed during CPA probably by binding of kallikrein, since purified CPA dial likrein blocked purified CĪINH activity. The antigen level of CĪINH was not affected by CPA.

This finding has diagnostic importance since false low levels of CĪINH activity may be detected in serum samples exposed to CPA favouring conditions. In fact this proved to be the case in a large proportion of subjects thought to be suffering from so-called acquired angioneurotic oedema (Quincke’s oedema urticaria) on ground of low CĪINH activity determined in a frozen and thawed serum sample. CĪINH activity proved to be completely normal in fresh samples.