bluuebunny/crossref_metadata_2025 huggingface dataset:
PROTEIN C, FIBRINOGEN KINETICS AND PLATELET SURVIVAL IN PATIENTS WITH RECURRENT VENOUS THROMBOSIS
Authors: Crow M J , Learoyd P A , Warrington R , Rajah S M
Published: June 2019

Recurrent venous thrombosis though uncommon leads to post phlebitic limb syndrome. The pathogenesis is not readily definable in all cases. We have examined the platelet, coagulation and fibrinolytic systems of a small group of patients with venographically proven recurrent thromboses. Currently 14 patients have been studied. Besides routine investigations all patients had the following: 111 In labelled platelet survival, 125 I fibrinogen kinetics, Protein C (functional and antigenic), a full fibrinolytic screen, antithrombin 111 and plasma B-Tg. With the exception of isotopic studies all investigations were performed on two occasions. No patient was suffering a thrombotic event at the time of inclusion into the study. Protein C levels, fibrinolytic screens and antithrombin 111 levels were within normal limits for all patients, while plasma B-Tg was raised. Platelet and fibrinogen kinetic data is shown in the table (mean+/−SEM):

Mean fibrinogen kinetics were normal with only 2 patients displaying increased turnover. Mean platelet survival was reduced and turnover raised, although a further 2 patients had normal values. These results indicate that this group of patients requires extensive investigation including isotopic studies. The shortened platelet survival time found appears to indicate platelet activation and consequently a possible therapeutic role for antiplatelet drugs combined with anticoagulants
PLASMA DEHYDROEPIANDROSTERONE SULPHATE CONCENTRATIONS AND DEEP VEIN THROMBOSIS AFTER MAJOR ABDOMINAL SURGERY
Authors: Mellbring G , Chotai J , Nilsson T K
Published: June 2019

Dehydroepiandrosterone sulphate (DHEAS) is a major secretory product of the human adrenal gland. Its precise functions are uncertain, but it has been postulated as a discriminator of life expectancy and aging. We have previously reported significantly lower plasma levels of DHEAS pre- and postoperatively in men developing DVT after major abdominal surgery, as compared to patients who remained free of DVT. Recent data suggest that the DHEAS concentration is also independently and inversely related to death from cardiovascular disease in men over 50 years of age. We here report data on preoperative plasma concentrations of DHEAS (measured with RIA) in 96 patients over 40 years of age, who underwent major abdominal surgery, and correlated the result to the development of postoperative DVT as diagnosed by the 125I-fibrinogen uptake test.

Thirty patients (31%) developed postoperative DVT during the first ten postoperative days. The plasma levels of DHEAS were significantly lower in the patients with postoperative DVT compared to those without (median 1.95 umol/1; Q1−Q3, 1.30−3.00 vs. median 3.35 umol/1; Q1−Q3 1.60−4.70; p < 0.02). Ninety per cent of the patients who developed postoperative DVT had a DHEAS value lower than 3.9 umol/1, while 45 % of the patients who remained free of DVT had a DHEAS value higher than 3.9 umol/1.

In conclusion, the data suggest that a low preoperative value of DHEAS predisposes the development of postoperative DVT in patients undergoing major abdominal surgery. It seems like the DHEAS concentration in plasma can be a valuable factor in a predictive index for postoperative DVT in those patients.
HAEMATOCRIT REDUCTION CONCOMITANT WITH ACUTE PHASE REACTANT RISE
Authors: Balnave K , Nelson S D , Moriarty A J
Published: June 2019

The study aimed to investigate haematocrit variation as acute phase reactants rise in myocardial infarction.

Serial measurements of haematocrit were undertaken in a cohort (N = 60) of patients over periods ranging from 6 to 28 days. Concurrent measurements of plasma fibrinogen were made by the method of Clauss. Patients with clinical or radiological evidence of heart failure were excluded as were patients with history or evidence of bleeding for any cause.

Results showed a small initial transient rise in haematocrit above baseline of 3% within the first 12 hours post-infarction, followed by a mean decrease below baseline of the order of −16%. The decrease was maintained at the time of discharge from hospital (mean 9.5 days) at a level of −11%. In the single case where serial haematocrits were measured up to 28 days, haematocrit was still reduced by −12%. The maximum individual percentage decrease measured in any patient exceeded −30% and occurred at 3 days post-infarct. The decrease parallelled the rise in plasma fibrinogen but apart from the early post−infarct period, the correlation was poor because of the rise in other acute phase reactants subsequently.

A postulated mechanism for the fall in haematocrit is that it reflects haemodilution. This is in part a physiological compensatory mechanism for the increasein colloid osmotic pressure that is attendant on the increase in acute phase reactants due to myocardial tissue destruction. The blood loss due to serial diagnostic phlebotomies cannot explain the magnitude of the decrease.
DO PLATELET INHIBITORS INCREASE OPERATIVE BLOOD LOSS?
Authors: Galvin DAJ , Meek A C , Pate P , McCollum C N
Published: June 2019

Although platelet inhibitory therapy improves arterial graft patency, surgeons are anxious that preoperative administration may increase operative bleeding. We investigated the effect of platelet inhibitors on blood loss during femoral artery replacement in dogs.

Thirty greyhounds were randomised to receive placebo, a thromboxane antagonist GR32191 25mg (Glaxo Group Research) or aspirin 150mg (ASA) plus dipyridamole 50mg (DPM) twice daily starting 48 hours prior to implanting a 6cm length of 6mm PTFE in the femoral artery using standardised incision, mobilisation and anastomosis with 6.0 prolene. All bleeding was collected in swabs which were then thoroughly washed in 2L heparinised saline. The erythrocytes were haemolysed by adding potassium cyanide and the haemoglobin concentration measured in a Coulter Haemoglobinometer (Coulter Electronics). Blood loss was calculated by comparison to 1:500 dilution of the same animal’s venous blood. The bleeding time of the arterial anastomosis was also recorded.

The mean (± sem) blood loss was similar in all three groups tending to be slightly less with GR32191 and ASA ± DPM at 135125 and 115±121 mis respectively, compared to 152±129 mis on placebo (NS). Anastomosis bleeding time appeared to be prolonged at 390±131 secs by the thromboxane antagonist compared to 291±40 with placebo and 224±136 with ASA and DPM, but this difference did not achieve statistical significance. There was a significant correlation (r=0.53) between blood loss and anastomotic bleeding time (p<0.001).

This method of measuring blood loss is easily applicable to patients and does not demonstrate any important tendency to increased bleeding with preoperative platelet inhibitors.
CLINICAL AND LABORATORY FEATURES ASSOCIATED WITH LUPUS ANTICOAGULANT
Authors: Cooper S M , Malia R G , Preston F E , Duncan S L B , Smith AR B , Greaves M
Published: June 2019

Recent evidence suggests a relationship between the presence of lupus anticoagulant and elevated titres of antibody to cardiolipin. Also an association with thrombotic tendency and with a high incidence of first trimester spontaneous abortion or second and third trimester fetal death has been noted. In order to investigate the relationship between anticardiolipin (aCL) antibodies and coagulation test abnormalities we have studied 12 subjects with systemic lupus erythematosus (SLE),10 with history of venous thrombosis, 30 with unexplained prolongation of clotting times and 34 with a poor obstetric history. Coagulation tests applied were: KCCT using two ’incomplete’ thromboplastin preparations and corrections with 20%/50% normal plasma; one-stage prothrombin time (OSP); dilute Russell’s viper venom time (DRVVT, including the platelet neutralisation procedure) and the heat stability test. IgG and IgM aCL antibody were assayed by enzyme-linked immunosorbent assay as described elsewhere. Abnormalities in more than one coagulation test were detected in 4 subjects with SLE (33%), 4 with thrombosis (40%), 6 with poor obstetric history (18%) and 10 with unexplained prolonged KCCT (33%). The DRVVT was most sensitive to the presence of lupus anticoagulant. Detectable aCL antibody was present in 12 subjects (IgG in 6, IgM in 4, both in 3), including some from each group. 2 had IgG aCL antibody and normal coagulation tests (1 SLE, 1 poor obstetric history). No coagulation or immunological abnormality was detected in 30 healthy non-pregnant and 13 healthy pregnant controls. Our data indicate the requirement for a comprehensive methodological approach for the detection of aCL antibodies/lupus anticoagulant and the clinical importance of such an approach.
FIBRIN DEPOSITS IN BONE MARROW AND CHANGES IN HAEMOPOIESIS AFTER ENDOTOXIN ADMINISTRATION
Authors: Pindur G , Seifried E , Rasche H
Published: June 2019

Thrombotic occlusion of microcirculation during DIC has already been studied in numerous organs, but little is known about analogous findings in the bone marrow. Therefore in rats under the influence of endotoxin the defibrination was examined for its relationship to changes inthe bone marrow and haemopoiesis. Bone marrow specimens were studied histologically by fibrin staining methods. Blood cells were measured automatically on the Coulter counter, fibrinogen by clotting assay. A fall in the thrombocyte and fibrinogen level was induced through a single injection of endotoxin with a maximum after 24 hours and 48 hours. In the same phase, after a short-term drop, a marked rise in the leukocyte count in the peripheral blood was observed. Following an initial increase the erythrocytes dropped and reached their lowest level after 48 hours. In the bone marrow 24 hours after endotoxin administration a large amount of fibrin deposits were observed in the small vessels. At the same time a clear reduction in all three haemopoietic cell lines was noticed. Between days five and ten the parameters of the peripheral blood normalized. Fibrin deposits in the bone marrow were no longer evident after three days. After 28 days an increase in the granulocytopoiesis and a continuing reduction in the megakaryopoiesis was observed.lt is concluded, due to the existence of fibrin deposits in the bone marrow in the beginning phase, that the observed haematologic alterations can not only be explained by the direct effect of endotoxin, but possibly also by the temporary microcircu-latory disturbances of the bone marrow during defibrination with its adverse effects on the haemopoiesis.
THE PROLONGATION OF THE THROMBOTEST CLOTTING TIME IN NEWBORNS
Authors: Hamulyák K , Devilée P P , Nieuwenhulzen W , Hemker H C
Published: June 2019

We investigated 40 human umbilical cord plasma samples of healthy full term infants. The samples were drawn under conditions which minimize in vitro activation of the haemostatic mechanism. In 67% clotting inhibiting material was present as judged from thrombotest dilution curves. The prothrombin (factor II) clotting activity (one stage method) ranged from 28 to 74% (mean value 50.6% ± 12.1). The correlation coefficient between the thrombotest clotting times and theprothrombin levels was -0.46. Using sensitive immuno assays for fibrin degradation products (XDP) and fibrinogen degradation products (FDP) based on monoclonal antibodies, we found that no degradation products could be demonstrated in the non-inhibited group (in the thrombotest dilution curves) whereas small amounts of these products were present in the inhibited group. These small amounts were undetectable using conventional assays. The most striking finding was the presence of fibrin and fibrinogen degradation products. The prolongation of the thrombotest clotting time could be imitated by adding small amounts of purified degradation product fragment X to umbilical cord plasma with a normal thrombotest clotting time. The thrombotest clotting time is often used in clinical practice to obtain an impression of the levels of the vitamin K-dependent coagulation factors. We conclude that the thrombotest clotting time is of limited value in the assessment of the vitamin K-dependent coagulation factors in umbilical cord plasma because in 67% of the cases a further ("false") prolongation of the thrombotest clotting time is caused by small amounts of fibrin(ogen) degradation products. As utmost care was taken to avoid proteolytic breakdown in vitro, our findings most likely reflect an enhanced fibrino(geno)lytic activity in umbilical cord plasma in vivo.
PLACENTAL-DERIVED PGI2 INHIBITS CORD PLATELET FUNCTION: POSSIBLE ROLE OF PGI2 IN THE TRANSIENT HYPOREACTIVITY OF NEWBORN PLATELETS
Authors: Landolfi R , De Cristofaro R , De Carolis S , Ciabattoni G , Bizzi B
Published: June 2019

Previous studies on newborn platelets hyporeactivity have been performed on cord blood. In this study we demonstrated that fresh cord platelet poor plasma (C-PPP) contains a labile antiaggrega-ting substance which, added to adult platelet rich plasma (PRP), is able to reverse ADP-induced platelet aggregation. Measurements of 6-Keto-prostaglandin (PG) Fl± levels in C-PPP obtained from10 different normal newborns gave anaverage value of 1050 ± 361(SD) pg/mL. Significantly lower levelsof this prostaglandin were found in plasma samples obtained from two newborns 2 hours after the birth (mean = 150 pg/mL) and in PPP of ten control adults (mean = 25 ± 34 pg/mL). In three newborns, platelet aggregation was studied using both C-PRP and PRP obtained 2 and 48 hours after the birth. A marked reductionof platelet response to ADP and collagen was evident in C-PRP. Such hyporeactivity was mild at 2 hours and absent in the third day of life. These results show that PGI2 inhibits cord platelets and might be the cause of a transient platelet hyporeactivity in the newborn. Finally we demonstrated that washed newborn platelets, compared to adult platelets, have a significant increase ofthe apparent affinity constant (Ka)for fibrinogen and that fetal and adult fibrinogen have similar Ka for platelets.
PRESENCE OF PEROXIDES IN PLASMA OF RATS TREATED BY ORAL CONTRACEPTIVE ENHANCING AGGREGATION OF NORMAL PLATELETS
Authors: CIAVATTI M , BLACHE D , RENAUD S
Published: June 2019

Platelets and plasma were prepared from normal and oral contraceptive (ethinyloestradiol + lynestrenol) treated female rats. Thombin and ADP-induced aggregation of normal platelets resuspended in incomplete Tyrode’s buffer were studied after being preincubated either in normal plasma (N.P) or in plasma from oral contraceptive (OC.P) treated animals. Aggregation was drastically enhanced in OC.P as compared to N.P (about 3 fold) after only 4 min preincubation at room tenperature. If normal platelets were pretreated by butylated hydroxytoluene (BHT) and incubation and aggregation performed as above, the aggregation of N.P and OC.P treated platelets were identical. The dose of BHT was chosen so as not to affect normal platelet aggregation. Similarly, when OC.P was pretreated with an active preparation of peroxidase (3 U/ml plasma) and reduced glutathione (2mM), the enhancement of aggregation of normal platelets preincubated in OC.P was completely abolished compared to platelets incubated in N.P. When platelets were incubated with N.P or OC.P pretreated by catalase, pi ate let-induced aggregation was still found to be potentiated in OC.P comparatively to N.P although the absolute extent of aggregation was decreased in both cases as compared to no catalase pretreatment of the plasmas. The peroxidized fatty acid fraction was isolated from N.P and OC.P and tested on the aggregation of normal platelets. A far greater potentiating effect on platelet aggregation was found with the peroxidized fraction extracted from OC.P comparatively to that from N.P. Malonaldehyde was also found increased in plasma from OC.P compared to N.P if measured very quickly after the sampling. From these data we conclude that the potentiating effect of O.CP on platelet behavior might be mediated by lipid hydroperoxide, rather than by hydrogen peroxide probably through oxydative injury to platelets.
CHANGES IN THE COAGULATION AND FIBRINOLYTIC SYSTEM DURING PREGNANCY
Authors: Woodhams B J , Candotti G , Kernoff P B A
Published: June 2019

Blood samples were collected from 17 volunteers between 8-14, 26-28 and 32-34 weeks of pregnancy. Control samples were collected from 12 non-pregnant female volunteers not using oral contraceptives. All samples were assayed for fibrinopeptide A (FPA), B beta 15-42 and for cross linked D-dimer fragments. A sample was collected for measurement of the in vitro rate of generation of fibrinopeptide A from whole blood (FPA-R).

These results are consistent with an increased activation of coagulation (increased FPA and shortened FPA-R) during normal pregnancy, which is compensated for by a concomitant rise in fibrinolytic activity (increased D-dimer and B beta 15-42). In 2 patients who miscarried and 2 patients who developed hypertension during pregnancy changes indicative of a non compensated hypercoagulable state could be seen. The study shows that the progress of pregnancy is associated with increased activation of coagulation and a concomitant rise in fibrin(ogen) degradation products. This data shows that a combination of these tests may be capable of detecting changes in the haemostatic balance that occur before the onset of clinical signs of gynaecological problems.
SELECTED HEMOSTASIS PARAMETERS IN PREGNANCY AND HYPERTENSION
Authors: Saleh A A , Farag A M , Bottoms S F , Mammen E F , Hosni M , Ali A
Published: June 2019

Patients with preeclampsia are believed to have a state of compensated DIC, and especially the differential diagnosis between preeclampsia and chronic hypertension with pregnancy can be difficult. Selected hemostasis parameters were analyzed in 50 women with preeclampsia (P), 50 matched normal pregnant women (N), 14 women with known hypertensionand pregnancy (CH) and 13 persons with known chronic hypertension and superimposed preeclampsia (CH + P). None of the patients had clinical evidence of DIC. Platelet counts, mean platelet volume, antithrombin III, α2 antiplasmin and fibrinogen activities and fibronectin were assayed. The following data were

These data, together with higherlevels of fibrinopeptide A, platelet factor 4, 3 thromboglobulin and D-dimer in the P group suggests increased intravascular coagulation in preeclampsia. Fibronectin levels were markedly elevated only in the patient groups with preeclampsia. Discriminant function analysis of FN values between the groups revealed a78% diagnostic accuracy for Palone and 74% accuracy for CH + P
REDUCTION OF THE ANTICOAGULANT ACTIVITY OF PROTEIN C AND PROTEIN S DURING THE POSTOPERATIVE PERIOD
Authors: Vigano D'Angelo S , Gilardoni F , Seveso M P , Marassi A , Mari G , D'Angelo A
Published: June 2019

Protein S circulates in plasma as free protein S and in complex with C4b-binding protein, an inhibitor of complement activation. Only free protein S functions as the cofactor for the anticoagulant and profibrinolytic effects of activated protein C. Since isolated reductions of protein C and protein S result in increased thrombotic risk, only measurement of both proteins permits comprehensive evaluation of the antithrombotic potential of the protein C system. No information is available on protein C and protein S functional levels during the postoperative period, an established prothrombotic condition. The plasma changes of protein C, protein S and C4b-binding protein were followed in 40 patients with no malignancy undergoing abdominal surgery. No significant change of protein C and protein S activi ty was observed following minor operations. After major surgery, protein C anticoagulant activity dropped to 80% of preoperative levels during the first postoperative week (p<0.00l). Significant increase of both total protein S antigen (110%, p< 0.01) and C4b-binding protein (130%, p<0.001) were observed after major surgery resulting in reduction of free protein S antigen to 86% of pre operative values (p<0.001). Protein S anticoagulant activity matched the changes of free protein S antigen.

Albeit transient and moderate, the observed reductions of both protein C and protein S may act synergistically to cause significant impairment of the antithrombotic potential during the postoperative period. The effect of heparin prophylaxis on protein C and protein S postoperative levels is currently under investigation.
PHYSIOLOGICAL COAGULATION INHIBITORS (PROTEIN S, PROTEIN C AND ANTITHROMBIN III) IN NORMAL PREGNANCY AND IN SEVERAL HYPERTENSION STATES IN PREGNANCY
Authors: Gilabert J , Fernández J A , Espana F , Aznar J , Estellés A
Published: June 2019

The protein C (PC) - protein S (PS) anticoagulant system and antithrorribin III (AT III) were evaluated in normal pregnancy (group A, n= 53), severe preeclampsia (group B, n= 15) and chronic hypertension with superinposed severe preeclampsia (group C, n= 18). Group A was classified according to the stage of pregnancy as 1st (n=9), 2nd (n=ll) and 3rd (n=33) trimester (tr). A control group comprised 21 normal, non-pregnant women who were non-users of oral contraceptives. In normal pregnancy a significant decrease in the level of free protein S was observed in the 2nd trimester of pregnancy and was sustained throughout the remaining months. The other coagulation inhibitors (protein C and AT III) undergo no significant changes during pregnancy and remain within the limits of normality. In cases of preeclampsia a significant decrease in protein C was observed. It was more evident in severe preeclarrpsia but was also found in chronic hypertension with superiirposed severe preeclanpsia when compared with the normal pregnancy group at similar gestational age. No statistically significant differences in protein S were found when the normal and pathological groups were compared. AT III decreased slightly in the severe preeclamptic group but the decrease was not significant.

The decrease in protein C and AT III levels in severe preeclanpsia could be related with the microthrombotic state that these patients may present. However, protein S, which decreases during normal pregnancy, seems to play no role in preeclanpsia.
INHERITED DEFICIENCY OF FUNCTIONAL PROTEIN S IN A JAPANESE FAMILY
Authors: Nakamura K , Iijima K , Inoue N , Nishioka J , Suzuki K
Published: June 2019

A family with a history of severe recurrent venous thromboembolic disease in some of the members was studied over three generations. In the propositus, a 36-year-old Japanese man, assays for the coagulation, anticoagulation and fibrinolytic factors including fibrinogen, plasminogen, antithrombin III and protein C associated with inherited venous thrombotic disease were normal. While markedly decreased protein S, a cofactor of activated protein C, was revealed. The functional protein S activity in plasma was lower than 5% of normal. The same laboratory data were shown in his paternal uncle and two sisters who had recurrent thrombotic episodes, and his asymptomatic father as well. The mean protein S antigen was 38%(range 22-52%) by the conventional Laurell rocket immunoelectrophoresis. But by the crossed immunoelectrophoresis and the Laurell rocket electrophoresis using the PEG 8000 precipitation method, the free form protein S was undetectable in their plasma(Table). Thus, a significant coincidence between the antigenic free protein S levels and the functional protein S activities was clarified.

It is suggested that the recurrent, thrombotic disease family is due to an inherited deficiency of protein S, larly of free form and functional protein S.
PROTEIN C : ROUEN - A NEW HEREDITARY PROTEIN C ABNORMALITY WITH LOW ANTICOAGULANT BUT NORMAL AMIDOLYTIC ACTIVITIES
Authors: Borg J Y , Vasse M , Monconduit M
Published: June 2019

During the last three years, we could detect hereditary quantitative protein C (PC) deficiency in 43 patients belonging to 18 families. In those defects type I without oral anticoagulant treatment, the values of PC measured either by an ELISA method (PC:Ag) or by a chronometric functional assay were very closed and well correlated. (Results expressed in % of normal pooled plasmas PC:Ag m=44,l %, SD = 15,3 ; PC : activity m = 49,5, SD = 13,5 - correlation r = 0,82). In a 32 year#old man with a severe thrombo-embolic disease and in 11 related people, we could diagnose a hereditary qualitative PC deficiency type II, because of a discrepancy between normal PC:Ag levels (m = 105 %, SD = 20,3, range = 78-143) and low PC anticoagulant activities (m = 46 %, SD = 9,5, range = 30-60). FunctionalpC studies included assays, with or without preliminary adsorption on baryum citrate or aluminium hydroxide, with various PC activators (thrombin, PROTAC venom), in chronometric and amidolytic assays.(Normal protein S levelswere first tested).

As shown by those results, PC activity is normal in amidolytic assays even after preliminary adsorption whatever the activation is. On the contrary, the PC anticoagulant activity is reduced in any technique. We can conclude that the activation is normal. Crossed immunoelectrophoresis (CIE) with or without calcium showed normal migration as compared to controls. Normal adsorption on insoluble salts and normal Ca-binding in CIE allow us to say that the abnormal PC is not completely acarboxylated. As amidolytic assays (normal in patients) do not assess the ability of activated PC to interact with protein S (PS) and phospholipids via calcium, 3 hypothesis can explain the functional abnormality:

- abnormal binding to PS

- abnormal binding to phospholipids due to partially carboxylated glutamic acids (which would be sufficient to promote adsorption)

- defective inhibition of Va and Villa because a conformational change allowing only hydrolysis of little synthetic peptides.
AMIDOLYTIC DETERMINATION OF ANTI-ACTIVATED PROTEIN C ACTIVITY IN PLASMA
Authors: Nicham F , Martinoli J L
Published: June 2019

Anti-activated protein C (anti-APC) potency of plasma was studied using purified bovine activated protein C (Bovine APC) and the chromogenic peptide substrate CBS 65.25. The choice of bovine instead of human APC was justified by a better sensitivity (Km = 0.14 and 0.42 mM respectively). Inhibition was shown to be dramatically enhanced by the presence of Heparin and calcium. No significant difference occurred for pH values up to 8.2 for both inhibition and hydrolysis reactions.

In the final test, O.l ml of 1:5 diluted plasma (Tris buffer saline, pH 8.4, containing 5 U/ml of Heparin) were incubated at 37°C with 0.2 ml of Bovine APC (0.125 U/ml). After 10 minutes of inhibition, 0.2 ml of CBS 65.25 (1.5 mM/1) were added to the mixture and the change in absorbance was recorded at 405 nm for 2 minutes. In these conditions linearity of the dose-response curve was ensured from O up to 130 % of activity (normal plasma pool being assigned to 100 %) ; day to day precision was 1.9 %. When a normal plasma was overloaded with different purified inhibitors such as antithrombin III, cl-esterase inactivator, alpha 2 macroglobulin, the measured anti-APC activities were not affected at all. It could be concluded that this test measures protein C inhibitor described by Suzuki.

Levels in 23 normal individuals averaged 97.7 %, giving a normal range of 77 - 118 %. Levels were below normal in 6 of 10 patients after surgery (54.1 +/- 4.8 %), in 18 of 19 patients with liver disease (49.5 +/- 9.6 %) and in 4 of 18 coumarin treated patients (54.9 +/- 6.5 %). In 9 of 10 patients previou sly characterized as type I protein C deficient, a statistically significant increase in anti-APC activity was observed (mean 110.7 +/- 7.7 %).

The use of a chromogenic peptide substrate has led to a sensitive and fast assay for anti-APC activity in plasma. That could be of interest in clinical investigations and knowledge of regulatory mechanisms in thrombotic disorders.
THE REGULATION OF FIBRINOGEN PRODUCTION INVOLVES AT LEAST ONE OTHER HEPATOCYTE GENE
Authors: Fowlkes P M , Lund P K , Blake M , Snouwaert J
Published: June 2019

It is currently thought that glucocorticosteriods have a direct effect on the transcription of the alpha, beta and gamma fibrinogen genes. However, our studies indicate that while corticosteriods play a role in fibrinogen production, this role is not due to transcriptional activation via glucocorticosteriod receptors. In initial experiments, we compared the levels of fibrinogen mRNA in hepatocytes isolated from hypophysectomized rats to those from control animals. The levels of mRNA in hypophysectomized rats, which produce little ACTH or corticosteriods, were significantly higher than the levels in control animals. Albumin mRNA levels were unaffected by hypophysectomy. These results are in opposition to those which we had anticipated. Based on previously published data, we had thought that physiologic deprivation of corticosteriods would lead to decreased levels of fibrinogen. We propose that these results are related to the negative feedback that corticosteroids have on Hepatocyte Stimulating Factor (HSF) production through a tightly controlled feedback circuit. To investigate the role of corticosteriods in fibrinogen gene regulation, we have conducted experiments with primary hepatocytes in culture and rat FAZA cells (continuous hepatoma cell line). There is a 4 to 5 fold increase in fibrinogen production when these cells are treated with HSF but no change when these cells are treated with dexamethasone alone. However, there is a marked additional increase in the production of fibrinogen with the combination of dexamethasone and HSF. Data gathered through kinetic analysis of this synergistic interaction suggest that the maximum response to HSF requires another gene product whose production is responsive to dexamethasone. Detailed analysis of the rate of transcription of thegamma fibrinogen gene, its processing and mRNA turnover suggests a specific role for this gene product in regulating fibrinogen synthesis. Characterization of this gene product will lead to greater understanding of the regulation of the Acute Phase Reactants.
The Synthesis of Factor V in Tissue Culture and Isolated Organ Perfusion
Authors: Giddings J. C. , Shaw E. , Tuddenham E. G. D. , Bloom A. L.
Published: June 2019

Human foetal tissues were maintained in tissue culture and the presence of factor V detected by an indirect immunofluorescence technique. Established monolayer cultures of human foetal liver cells showed positive immunofluorescence when treated with rabbit anti human factor V serum and fluorescein isothiocyanate conjugated with anti rabbit immunoglobulin. Parallel liver cultures containing cycloheximide did not show positive fluorescence when stained by the same technique.

Hepatic factor V synthesis was also investigated by an ex-vivo organ perfusion method using adult rat livers. Factor V synthesis was most clearly demonstrated utilising leucocyte-and platelet-poor perfusate. Factor V synthesis was totally inhibited by cycloheximide and was inhibited by actinomycin D after an initial delay. Factor V synthesis was not inhibited by Warfarin.

The results indicate that factor V is synthesised in the liver by a mechanism similar to that of other plasma proteins and is not dependant on vitamin K for its activity.
Immunological Method for the Detection of the Carrier of Haemophilia B.
Authors: Matsuoka M. , Ito M. , Sakuragawa N. , Takahashi K.
Published: June 2019

Both the immunoassay and bioassay were performed on factor IX activity of haemophilia B patient. Their values were compared with each other.

The immunoassay by neutralization was performed as follows: antibody to factor IX was obtained by immunization of purified factor IX to rabbit which was isolated by the technique of DEAE-Sephadex column chromatography using eluate from BaS04 which absorbed factor IX of normal human plasma.

In approximately 90% of the cases of definite carriers of haemophilia B, the activity of factor IX by bioassay was observed to be lower than that of factor VIII of haemophilia A carrier. The factor IX activity was observed to be at the same level as factor IX antigen by immunoassay in almost all of the cases, but in the cases of the mother of haemophilia B, and the North Carolina type of haemophilia B the factor IX antigen was much greater than that of activity by bioassay. The same results were obtained by the above mentioned methods using inhibitor substance arising from severe haemophilia B patient.

It was suggested that the immunoassay method is useful in detecting the carrier of haemophilia B and North Carolina type of haemophilia B.
Fibrinogen Biosynthesis
Authors: Blombach B. , Redman C. , Kudryk B.
Published: June 2019

A mixture of 3H-labeled amino acids was injected into dogs. Blood was collected at specific time intervals and fibrinogen from each corresponding plasma fraction was isolated by affinity chromatography. After reduction and alkylation, the fibrinogen chains were separated either by CM-cellulose chromatography or preparative SDS-gel electrophoresis. In one dog whose fibrinogen level was approximately 8 mg/ml, it was found that both the γ and B β chains had lower radioactivity when compared to the Aα chain, at least in the early times of appearance in blood. When this experiment was repeated in another dog whose fibrinogen level was 2 mg/ml and non-labeled amino acids were given as “chaser”, the radioactivity in the γ and Bβ chain did not reach the same value as that in the Aα chain, even after a three hour period. The significance of these findings with respect to rate of synthesis, secretion and assembly of the polypeptide chains will be discussed.

Experiments were also performed using dog liver slices incubated with radioactive amino acids. Labeled, fibrinogen-related, material from detergent-solubilized rough endoplasmic reticulum (HER) could be recovered either by affinity chromatography or immunoprecipitation. Fractions were also isolated from smooth endoplasmic reticulum and Golgi. Analysis of the material isolated from BEB suggests that it may not consist of all three chains but rather one or possibly two of them to be either in degraded (during isolation procedures) or precursor form.

(Supported by N.I.H. grants HL 09011–11 and AM 13620.)
Observations on Fragment D Species Arising During Hydrolysis of Crosslinked Human Fibrin
Authors: Mosesson M. W. , Finlayson J. S.
Published: June 2019

A covalently linked bimolecular form of plasmic Fragment D has been identified by several investigators in digests of F. XIIIa-crosslinked (xlinked) fibrin (f). Peptides containing xlinks between α chains separate from the clot matrix prior to release of D ; therefore, such multimolecular core fragments must be joined by xlinked remnants of γ chain dimers. This observation has been interpreted as evidence for the so-called ‘mono-meric D’ fibrinogen structural model (Marder et al. J. Biol. Chem. 244, 2111 1969,). For this interpretation to be unambiguous, virtually all xlinked γ regions must retain their continuity with the D moiety at the time of its release from the clot. The studies to be reported did not support that presumption, and thus the observation by itself is consistent as well with the ‘dimeric D’ structural model (Mosesson et al. J. Biol. Chem. 1973, 248, 7913).

Human f was xlinked by F. XIIIa to achieve at least complete γ dimerization. Following syneresis, clots were transferred to EDTA-containing buffers (pH 8.6), plasmin was added, and samples of the buffer solution were examined by dodecyl sulfate gel electrophoresis. Monomolecular species of D were generated as were derivatives corresponding to multi-molecular xlinked forms of D. Ultimately, although xlinked sites are themselves refractory to plasmic hydrolysis, all xlinked forms of D were degraded to species indistinguishable from those arising in digests of fibrinogen or non-xlinked f. These results indicate that intermolecular connections created by introduction of xlinks between γ chains are severed during the plasmin-catalyzed formation of D fragments. This event may prove to be a requirement for the release of D from clot matrix.

(Supported by NHLI grant HL-11409.)
Early Endothelial Injury
Authors: Lough J. , Moore S.
Published: June 2019

A variety of blood constituents were injected into an isolated segment of rabbit aorta to determine which elements might be involved in early endothelial injury. Test materials consisted of platelet rich plasma (P. R. P.) alone; P. B. P. plus adenosine diphosphate (A. D. P.); P. R. P. plus tendon extract; P. B. P. plus thrombin; ultrasonicated P. R. P. alone; platepoor plasma (P. P. P.) alone and thrombin in saline. Each experimental mixture was left in the aorta for 15 minutes, followed by reflow for 20 min. The vessel was then flex by glutaraldehyde perfusion. Thick sections of the entire circumference of the aorta were taken for phase contrast microscopy and representative areas were selected for electron microscopy. In control experiments and with sonicated P. R. P. and P. R. P. plus tendon extract, the endothelium was normal. With P. R. P. alone, P. P. P. alone and with P. R. P. plus A. D. P. there were occasional subendothelial vesicles. When P. R. P. plus thrombin and P. P. P. plus thrombin were injected separately to form a thrombus or when thrombin in saline was used, there was extensive subendothelial vesiculation with focal ulceration and adherence of thrombus to endothelium. Severe injury was associated with the presence of thrombin initiating the polymerization of fibrinogen to fibrin. Electron micrographs demonstrate the earliest lesion as a disruption of the superficial fibrillary elastica with separation of overlying endothelium.

Additional Key Words: Plasminogen activation, turbulent flow, atherosclerosis, encrustation.
Mechanism of Collagen: Glucosyl-transferase and its Relation to Platelet: Collagen Adhesion
Authors: Smith D. F. , Kosow D. P. , Jamieson G. A.
Published: June 2019

Elucidation of the enzymatic mechanism of collagen: glucosyltransferase is essential to an understanding of its role in platelet function. A soluble form of the enzyme has been purified 100-fold and a sensitive new assay system developed. Studies with effectors such as UDP, ADP and ristocetin under steady state conditions have shown that only two of the possible sequential mechanisms are consistent with the kinetic data. Inhibition by UDP and ADP is competitive with UDPG but non-competitive with galactosylhydroxylysine. They would not, therefore, be expected to inhibit the formation of an enzyme-substrate complex with collagen. Under physiological conditions, their presence would be expected to increase the affinity of the cell surface enzyme for its acceptor on collagen in the case of the ordered mechanism, or not to affect it in the case of the random mechanism. These data are consistent with the potentiation of collagen-induced aggregation by ADP, and the lack of effect of UDP on the adherence of platelets to collagen.

(Supported, in part, by USPHS.)
In Vitro Studies on Thrombolysis with Plasminogen and Streptokinase
Authors: Rice Lory R. , Sogar S. , Kakkar V. V.
Published: June 2019

It has been suggested that in vivo thrombi and emboli become deficient in intrinsic plasminogen (Pig) though plasma proteins are still able to percolate through the fibrin mesh which forms the basic structure of the clot. Thus it is reasonable to expect that such Pig-deficient thrombi will be degraded at a faster rate, if perfusion with a plasminogen activator is preceded by a preliminary perfusion of plasminogen itself. Such a hypothesis has important implications for clinical therapy.

Our studies were undertaken in vitro, using human thrombo-emboli removed at postmortem. An initial perfusion wit 125I-labelled Pig showed an uptake of Pig into the thrombus about 3 times higher than the perfusion fluid, as shown previously (Strachan et al., 1974). Subsequent perfusion with buffer containing streptokinase (Sk) showed increased lysis under certain conditions, compared to that when either Pig was omitted from the preliminary perfusion or Sk from the main perfusion. This enhanced lysis was shown to depend upon the Pig concentration of the perfusion fluid.

Strachan, C. J. L. et al. (1974), Thrombosis Res., 4, 303.
Purification of Human Urokinase and its Topographical Localisation in Renal Parenchyma
Authors: Andrassy K. , Ritz E. , Bleyl U. , Egbring R.
Published: June 2019

Urokinase Leo was separated by agar zone electrophoresis into an anodic and cathodic fraction. The cathodic fraction, isolated from agar gel by ultracentrifugation, showed two precipitation bands with rabbit Urokinase antibodies. Band I displayed main Urokinase activity, in band II Urokinase was present in a high molecular weight complex with human serum proteins (albumin, a2-macroglobulin, a2HS glycoprotein); with affinity chromatography further separation of Urokinase isoenzymes from serum proteins was possible. The isoelectric point of these two Urokinase isoenzymes were pH 6.8 and pH 8.7 respectively in preliminary results with isoelectric focusing. Purification steps were controlled by disc gel electrophoresis and immunological techniques (Ouchterlony technique, Immunoelectrophoresis, clot lysis test with Urokinase antibodies).

Topographic localisation of Urokinase in renal tissue, investigated with antibodies against Urokinase isoenzymes, revealed Urokinase activity both in the iuxtamedullary region (V. arcuatae, V. interlobulares, less V. recta) and in calyceal epithelia of the renal pelvis.
Fibrinogen/Fibrin Degradation Products in Defibrination Syndrome Analysed on the Basis of Molecular Weight on Gel Exclusion Chromatography
Authors: Kazama M. , Abe T.
Published: June 2019

1) Fresh citrated plasmas of cases of defibrination syndrome were chromatographed on Biogel A-5 m column, which revealed a two-peaked elution profile of fibrinogen-related antigen with reproducibility. The first peak was eluted at the Vo of the column and the second one at Ve of the early FDP. It was found in the preliminary experiments in vitro, that the big molecular FDP was formed exclusively in the process of fibrin clot lysis, irrespective if it was non-stabilized or stabilized.

2) FDP in thesen patients’ serums, after thorough digestion with plasmin, were chromatographed on Sephadex G200 column. A reproducible elution profile of FDP-D antigen revealed single peak, of which Ve was corresponded with that of FDP-D monomer derived from fibrinogen or non-stabilized fibrin.

It was postulated that the FDP in plasmas of defibrination syndrome were mainly originated from the fibrinogen or non-stabilized fibrin formed in the process of the diseases.
Fibrinogenolysis: Studies on its Degradation Products
Authors: Bouvier J. , Braude R. , Altman R.
Published: June 2019

Human fibrinogen is degradated by plasmin into four generically accepted major products namely X, Y, D and E, and three lower molecular wheight fragments called A, B and C. A new early fragment named E’ is described when plasmin degradation of fibrinogen was analysed in agar and poliacrylamide electrophoresis, Sheidegger inmunoelectrophoresis and Laurell cross antigen-antibody electrophoresis. In the most early moment of proteolysis the electrophoretic position of this fragment is similar to there of fibrinogen X and Y fragments, but it migrate faster toward the anode when further digests were analysed. It seem to appear together of fragment X and, after loosing small peptides, will finally reach in the gel, the terminal fragment E position. On this basis, we suggest that from each fibrinogen molecule derivate two fragments E; one released from fragment Y and the other from the fragment E’ here described. Some modifications of the fibrinogen degradation scheme are postulated.
Drug Inhibition of Ristocetin-induced Platelet Aggregation
Authors: Tann G. , Flute P. T.
Published: June 2019

Administration of aspirin or phenylbutazone to normal subjects inhibits ristocetin-induced platelet aggregation in their platelet-rich plasma for periods up to two weeks. Two patterns of response have been observed, a complete lack of aggregation or an absence of secondary aggregation. Either can be overcome by increased amounts of ristocetin. Gel-filtered and albumin-density gradient washed platelets have also been investigated. A similar but transient inhibition has also been observed in some normal subjects due to an unknown cause. These results are important when considering the diagnosis of von Willebrand’s disease.
Response of Normal and Pathological Platelets to Heparin Added in Vitro and in Vivo
Authors: David J. L. , Herion F. P.
Published: June 2019

Heparin one unit/ml added in vitro to normal citrated PRP increases the platelet aggregation induced by ADP or Adrenaline with regard to aggregation measured in citrated PRP without heparin. This phenomenon is accompanied by an increase of the release reaction of 14C-5HT, ATP and ADP. In the same experimental conditions, pathological platelets (“aspirin-like syndrome”) respond by an increased aggregation without release reaction. This effect of heparin on normal or pathological platelet aggregation is inhibited by ASA or by Dipyridamole. – In citrated PRP obtained from blood of heparinized normal subjects, platelet aggregation and release reaction of platelets are increased with regard to their response tested before heparinization. Addition of ASA or Dipyridamole in PRP inhibits the effects of heparinization. – In two subjects with a platelet disease and a mild bleeding tendency, heparinization in vivo does not lengthen the bleeding time but increases the aggregation, without any release reaction. In vitro addition of ASA or Dipyridamole inhibits the increase of aggregation observed in absence of release reaction. -Besides their theoretical aspects concerning the mechanism of inhibition of platelet aggregation by ASA and Dipyridamole, these observations suggest the possible benefit of antiaggregant drugs for subjects treated by heparin.
Morphology of Haemostatic Plugs Before and After Aspirin
Authors: Wester J. , Veen J. van der , Sixma J. J.
Published: June 2019

Punch biopsies of bleeding time wounds according to Mielke were taken at 3 minutes in normal volunteers before and 2.5 hours after 2 gr. of aspirin. Haemostatic plugs were found at the end of blood vessels. No plug was encountered that had no relation to a blood vessel. Fibrin on the other hand was found in the wound but not in the blood vessel. Some vessels were occluded by capsule-like haemostatic plugs. In many others an extension into the blood vessel was observed. The extension into the blood vessel showed little or no degranulation. Blood platelets were more degranulated and showed peripheral ballooning before aspirin intake. After aspirin intake much less degranulation was observed and less peripheral ballooning was found.
Photometric Assay of Antithrombin III with a Chromogenic Substrate
Authors: Vinazzer H.
Published: June 2019

The synthetic substance Benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) is a sensitive substrate for thrombin which splits off p-nitroaniline from the tripeptide molecule. The yellowish colour of the split produkt can be measured photometrically at an optimal wavelength of 405 nm. Inactivation of thrombin by antithrombin III results in inhibition of this reaction. There is a linear relation between the percentage of thrombin inhibited and the activity of antithrombin III. As a result of test series on various modifications of the method an optimated procedure is described. The standard error of the assay was calculated at 3,2 per cent. Comparable results could be obtained between the coagulation method of Hensen and Loeliger and the chromogenic substrate method when both assays were carried out simultaneously on 18 patients as well as on pooled standard plasma.