bluuebunny/crossref_metadata_2025 huggingface dataset:
FIBRONECTIN, ANTITHROMBIN III, VON WILLEBRAND-FACTOR-ANTIGEN AND THROMBOCYTES DURING SEPTICEMIA, PNEUMONIA, SHOCK AND POLYTRAUMA
Authors: Ziemen M , Fierek M , Francos A , Si nn J , Breddin K
Published: June 2019

In 56 patients with septicemia (N=10), septic shock (N=10), cardiogenic shock (N=7), bacterial pneumonia (N=6) and polytraumatized patients (N=23) fibronectin, von Wi11ebrand-factor-anti gen (vWF:AG), fibrinogen, antithrombin III (AT III) and platelet count were measured during the first week of illness. Like fibronectin vWF:AG is synthesized in the endothelial cells and is a mediator for platelet adhesion. Decrease of AT III and platelet count are sensitive indicators if activation of haemostasis

In all patients with septic shock on the first day of illness fibronectin was decreased (12.9ଗ.2mg/dl; mean +standard deviation) as well as AI III (55넠%) and platelet count (129 000녶 000/mm ). All patients with bacterial pneumonia showed lowered fibronectin levels on Day 1 (12.2±8.5mg/dl), at the same time AT III and platelet counts were lowered in most of these patients. On the first day of septicemia or polytrauma a slight decrease of fibronectin, AT III and platelet counts were observed: the mean of each of these parameters was below the normal range. During cardiogenic shock fibronectin and platelet counts were normal in all patients. vWF:AG and fibrinogen were elevated in nearly all groups during follow up; 1 week after cardiogenic shock the mean of vWF:AG exceeded 400%

In contrast to vWF:AG and fibrinogen there was obviously a similar behaviour of fibronectin, platelet count and antithrombin III, possibly as signs of activated haemostasis. vWF:AG and fibrinogen seem to react independently, no correlation was found between these parameters and fibronectin in severely ill patients
ANTITHROMBIN III SUBSTITUTION IN EXPERIMENTAL DIC INDUCED BY ENDOTOXEMIA
Authors: Miyakawa T , Farag A M , Assarian G S , Dawe E J , Mammen E F
Published: June 2019

Antithrombin III (AT) levels decrease in patients and certain animal species during disseminated intravascular coagulation (DIC), and substitution of AT with concentrates has been studied in humans as well as some animals with generally favorable results. Based on previous positive data (Semin. Thromb. Hemost. 11:373, 1985) in a canine DIC model, acidosis induced, we elected to study the effect of human AT concentrates on a canine endotoxin (ET) DIC model. Endotoxin (Difco) was administered i.v. as a 0.5 pg/kg bolus, followed, 19 hours later, by a 3 hour i.v. endotoxin infusion of 0.5 mg/ kg/hour (n = 7). Hemodynamic parameters (blood pressure, cardiac output) and hemostasis values (platelet counts (PC), fibrinogen (Fb), antithrombin III (AT), a2 antiplasmin (AP), fibrin(ogen) split products (FSP) levels and protamine sulfate tests (PST)) as well as oncotic pressures were measured before and 7 times over 5 hours during and after ET infusion. No significant hemodynamic alterations were noted, but PC dropped by about 50%, AT and AP by 40-50% while FSP increased markedly and PST became positive. Oncotic pressures dropped by 30%. Five animals received a bolus of 50 U/kg human AT concentrate (Behring) 30 minutes after initiation of the ET infusion. The obvious rise in AT activity was associated with higher AP levels, significantly lower FSP levels and less positive PST. Increasing the dose of AT to 100 U/kg (n = 3) had an even greater effect on AP, FSP and PST. The administration of AT (50 U/kg) before the endotoxin infusion (n = 2) led to no rise in FSP, no change in AP and an only slight increase in PST positivity in one of the two animals. Based on these preliminary data we conclude that AT substitution in this canine endotoxin model seems to positively influence hemostasis changes suggestive of DIC
THE DOSE DEPENDENCE OF ENDOTOXIN INDUCED ACTIVATION OF THE PLASMA CONTACT SYSTEM. AN IN VITRO STUDY
Authors: Røise O , Bouma B N , Stadaas J O , Aasen A O
Published: June 2019

The dose and time dependence of endotoxin induced activation of the plasma contact system was studied in citrated plasma incubated at 37,4 °C. During the first six hours no significant changes were seen. At 12 hours markedly elevated plasma kalli-krein (KK) activity was found in plasma tested with the highest doses of endotoxin (2000 and 200 γg/ml). Corresponding to the elevated KK activity reductions of both plasma prekallekrein (PKK) and functional kallekrein inhibition (KKI) were seen. Hageman factor (FXII) values determined immunologically were also reduced parallel to the decreases of PKK and KKI. Changes in high molecular weight kininogen (HMwK) values were seen as well

In plasma tested with doses of endotoxin lower than 200γg/ml no sign of activation was seen at 12 hours. At 24 hours, however, increased KK activity and decreased KKI and PKK values were seen in plasma incubated with 20 γg/ml of endotoxin. In test plasma with endotoxin concentrations of 2 and 0,6 γg/ml we found . sign of activation in 7 of 8 test tubes using 2 γg/ml and in 2 of 6 test tubes using 0,6γg/ml. In test plasma with the lowest dose of endotoxin (2,0 γg/ml) and in control plasma no changes in either KK activity, KKI, PKK, FXII or HMwK values were found at 24 hours

The study shows thaf- endotoxin induced activation of the contact system in citrated plasma is a slow process which is both time and dose
ENDOTOXIC LIPID A INDUCES BINDING OF FIBRINOGEN TO HUMAN PLATELETS VIA PROTEIN KINASE C PATHWAY
Authors: Timmons Sheila , Grabarek Jadwiqa , Hawiqer Jack
Published: June 2019

Endotoxic Lipid A is the biologically active principle of lipopolysaccharide of Gram-negative bacteria, a most frequent cause of sepsis underlying Disseminated Intravascular Coagulation (DIC) and shock. We have shown that endotoxic Lipid A activates Protein Kinase C in human platelets. Phosphorylation of a 47kDa protein (P47), a marker for Protein Kinase C activation, was observed within the first minute of interaction of Lipid A with platelets. This was accompanied by gradual exposure of the receptor for 125I-labeled fibrinogen (F). Binding of 125I-F was saturable and specific. When Lipid X, a precursor of endotoxic Lipid A and its competitive inhibitor, was used, the binding of 125I-F was blocked with 50% inhibition at a 1:1 stoichiometry between Lipid X and Lipid A. At the same time, phosphorylation of P47 was prevented. Since Lipid X constitutes a "half molecule" of Lipid A, we interpret these results as indicative of competitive blocking of endotoxic Lipid A in terms of Protein Kinase C activation and exposure of platelet receptors for fibrinogen. Binding of fibrinogen is necessary for platelet aggregation and endotoxic Lipid A-induced aggregation was also blocked by Lipid X. Endotoxic Lipid A-induced exposure of fibrinogen receptors via the Protein Kinase C pathway can contribute to involvement of platelets in microcirculatory thrombosis observed in patients with DIC and Gram-negative sepsis
HUMAN PERIPHERAL MONOCYTES STIMULATED WITH ENDOTOXIN IN VITRO SHOWED A FAST PROCOAGULANT ACTIVITY (PCA) ELEVATION AND A LATE ANTIFIBRINOLYTIC RESPONSE
Authors: Engebretsen L F , Kierulf P , Lyberg T , Andersen A B , Gulla M , Joo G B
Published: June 2019

Septicemia causes severe disseminated intravascular coagulation (DIC). Monocytes may contribute to the DIC process through endotoxin stimulated synthesis of PCA. This study was initiated to evaluate the role of the monocytes in DIC further. Blood was taken from healthy persons. Mononuclear leucocytes (30?o monocytes) in microtiter wells (25.000 monocytes/well) were added 1 ug/ml endotoxin, and kept in culture from 0 to 160 hours. The cells were harvested, and the PCA (thromboplastin), the fibrinolytic activity (urokinase-PA), and the antifibrinolytic response (plasminogen activator inhibitor-PAI) were measured in the cells. Cell activities were assayed with chromogenic peptide substrates

In the stimulated cells the PCA dominated initially, whilst the antifibrinolytic activity was dramatically elevated after about 40 hours. The fibrinolytic response was minimal compared to the rise in the PAI activity

In conclusion, the monocytes favoured DIC development during the 160 hours observation period in our experiment through coagulation factor synthesis and hampered fibrinolysis. This support the hypothesis that the monocytes participate in the DIC process
CHROMOGENIC ENDOTOXIN ASSAYS. - FURTHER METHOD DEVELOPMENT AND PRACTICAL EXPERIENCE
Authors: Nilsson K , Friberqer P
Published: June 2019

A kit for the determination of endotoxin by using Lumulus lysate (LAL) and a chromogenic substrate has now been available for three years. Here we will summarize the present status concerning its usefulness in the control of parenteral drugs, in hygienic control and in clinical diagnosis.

The wide variation of uses, demands considerable work for method adaptation to the various types of specimen and laboratory. Almost all samples have to be pretreated to become compatible with the reagents in the assay. It has also been found to be important to consider variousdemands depending on the number of samples, how fast the answer is wanted, simplicity and accuracy of the assay that is needed as well as equipment available. In some cases a single screening test is required. Then a tube with 100 pi of LAL reagent can be taken from the freezer. The other extreme is the determination of hundreds of samples in each series. Then microplates and suitable readers can be of great help. Several other procedures used on special occasions allowing e.g. wide assay range or low reagent cost will also be presented.

Manual and microplate procedures have been found suitable and can be made safe regarding contamination and losses of endotoxin. Single stage and micro methods have been tested and found to work. Data concerning material and laboratory equipment have been collected and are now available.

By using a quantitative procedure forthe determination of the small amounts of endotoxin present in most samples, it has been possible to find optimal reaction conditions and to trace various kindsof errors. Procedures for method controland trouble shooting are also suggested.
PROTEIN C AND AT III IN ESSENTIAL HYPERTENSION
Authors: Bielawiec M , Kloczko J , Wojtukiewicz M , Borowska M
Published: June 2019

Since hypertension is generally accepted as a risk factor for atherosclerosis and, on the other hand, alterations in haemostasis were reported in the disease, we focused our interest on two plasma inhibitors-protein C and antithrombin III, which play critical roles in the regulation of blood clotting process. We studied 19 patients with newly diagnosed essential hypertension at ist and 2ncl degreeof the disease (acc.to WHO criteria). Protein C antigen level was measured by means of ELISA Protein C test (Boehringer Mannheim GmbH).

AT III and Factor VIII R:Ag concentrations were measured by rocket immunoelectrophoresis acc.to Laurell using monospecific antisera (Behringwerke AG, Marburg). In comparison with healthy subjects the patients with essential hypertension revealed statistically significant decrease in protein C antigen level (79 t 21% vs 101 ± 16; p≺0.01) and substantially increased F. VIII R: Ag values (174 ± 72% vs 103 ± 33%; p p≺0.01). No significant correlation was found between F. VIII R:Ag and protein C level. The patients had also elevation of AT III concentration ofno statistical significance. Since F. VIII R:Ag is produced and released by endothelial cells its elevated level in patients with hypertension may reflect injuryto the vascular wall. Decreased levels of protein C probably due to its increased consumption observed in the patients may contribute to imbalance in the haemostatic system and promote vascular complications.
PATIENTS WITH MILD ESSENTIAL HYPERTENSION HAVE INCREASED PLATELET SIZE AND RELEASE REACTION ANO SHOW INCREASED RECEPTOR RESPONSE TO INFUSED ADRENALINE
Authors: Lande K , Kieldsen I O S S E , Westheim A , Aakesson I , Hlermann I , Eide I , GiesdaL K
Published: June 2019

Hypertensive (n = 35) and normotensive {n = 44) men all 42 years old were studied. The hypertensive (HT) had larger venous platelets than the normotensive (HT) (7.46 ± 0.10 vs 7.12 ± 0.09 10-15 1, p = 0.01). Plasma concentration of |3-thromboglobulin (BTG) was increased in arterial blood in hypertensive (40 ± 8 vs 21 ± 2 ug/1, p=0.02) while the venous values were similar in the two groups. Despite similar sampling procedure, the normotensive subjects had markedly higher BTGconcentration in venous compared to arterial blood (p≺0.01) at variance from thehypertensive where the arteriovenous difference in plasma BTG concentration was not present. Adrenaline was infused to 13 hypertensive and 12 normotensive subjects with dose gradually increasing to 0.04 pg/kg/min. Forearm blood flow was measured by strain gauge technique and relative forearm resistance calculated as mean blood pressure divided by flow. Twelve normotensive subjects (control group) received saline infusion.

Change in forearm resistance reflectsβ2- activation of smooth vascular cells, change in platelet count reflects splenic liberation of platelets in response to adrenergic stimulation while change in BTG may reflect platelet release upon stimulation of α2 -receptors. Thus, middle aged men with essential hypertension show increased sensitivity to adrenaline infusion in vascular smooth muscle, spleen and platelets.
EFFECTS OF VITAMIN K ON VITAMIN K DEPENDENT PROTEINS IN NEWBORN INFANTS
Authors: YAMADA K , MEGURO T , SHIRAHATA A , NAKAMURA T , ASAKURA A
Published: June 2019

Plasma levels of vitamin K (VK) and VK dependent proteins ( factor E, factor VH, factor X, protein C and osteocalcin)were determined before and after VK administration to 22 newborn infants. Vitamin K2 syrup ( 2 mg/kg of body weight ) was orally administered to 9 healthy premature, 11 high risk and 2 VK deficient infants under 3 days of age. VK families extracted from plasma were separated by high performance liquid chromatography using a Cosmosil 5 Ci8 column, and separated VK families were detected by a fluorometry after their reaction with ethanolic sodium borohydride in a reaction coil connected by one-line to a chromatographic column. Total activity of factor E, factor VE and factor X was assayed by a Normotest ( Nyegaard ), and protein C was measured by protac/APTT and protac/chromogenic substrate ( S-2366 ) functional assay system ( American Diagnostica ). Osteocalcin levels were assayed by using of a RIA method before and after the absorption of plasma by hydroxyapatite.

After VK administration, plasma VK2 ( menaquinone-4 ) content increased from levels less than 0.012yg/ml to levels between 15.9 and 70.9μg/ml, excluding one case in whom plasma VK was not detected after VK administration. Compared with Normotest values and osteocalcin levels of age-matched healthy newborn infants treated without VK, premature, high risk and VK deficient infant levels significantly increased after 24 hrs and after 7 days of VK administration. No correlation was seen between the increase of plasma VK contents and the increase of Normotest values after VK administration. On the other hand, no significant increase of protein C assayed by both methods was observed in healthy premature and high risk infants after VK administration.

These results indicate that the change of protein C after VK treatment is different from that of factor II, VII, X and osteocalcin.
α2-MACROGLOBULIN IS A MORE IMPORTANT INHIBITOR OF THROMBIN IN INFANT PLASMA THAN IN ADULT PLASMA
Authors: Fernandez F , Schmidt B , Andrew M , Ofosu F A
Published: June 2019

The concentrations of the three major inhibitors of thrombin (IIa) differ significantly in adult and infant plasma. The extent to which these differences contribute to the rates and profiles of IIa inhibition in infant and adult plasma is unknown. We determined this by adding 2 NIH U/mL of I-human α-IIa to anequal volume of defibrinated plasma for 30s at 37°C. After SDS�PAGE and autoradiography, free Ila and complexes of IIa with antithrombin III (IIa-ATIII), heparin cofactor II (IIa-HCII) and α2-macroglobulin (αa-α2M) were quantitated in 3 types of pooled plasmas: cord; 6 month old infant and adult. Thrombin was inhibited more slowly incord plasma than in adult plasma (p‹0.001). in addition, while ATIII was the major inhibitor of Ila in adult plasma, ouM was equally as important as ATI 11 in cord and infant plasmas (p‹0.001). When cord plasma was supplemented with purified ATIII, the extents and profile of Ila inhibition by cord and adult plasmas were indistinguishable. The addition of heparin (0.2U/mL) accelerated the inhibition of Ila by all 3 plasmas, with ATIII the predominant inhibitor. Thus, 83% of the inactive Ila was bound to ATIII in adult plasma, 77% in each of cord and 6 month old infant plasmas. However, significantly more Ila was inactivated by adult plasma than the other two plasmas. These results suggest that cord plasmas are intrinsically less able to inactivate Ila than adult plasma. While the overall ability of the plasma of 6 month old infants to inactive Ila is comparable to adult plasma, the profile of inhibition of Ila in the former, both in the presence and absence of heparin, is more closely related to neonatal than adult plasma, likely reflecting high αM levels.
PREDICTIVE VALUE OF LOW AT III LEVELS FOR THE OCCURRENCE OF IVH, IRDS AND DEATH IN PREMATURE NEONATES
Authors: van den Berg W , Peters M , Breederveld C , ten Cate J W , Koppe J G
Published: June 2019

The observation of AT III deficiency in premature neonates with Idiopathic Respiratory Distress Syndrome (IRDS), suggests a positive predictive value for a poor outcome. The underlying diffuse intravascular coagulation could generate serious hemorrhagic complications like Peri/Intraventricular Hemorrhage (IVH).

A prospective study was performed in consecutively born neonates to assess the predictive value of low AT III for theoccurrence of IVH, (gr. III/IV), IRDS, and death. Eighty-one neonates were included in the study during a period of 5 months. AT III levels were determined immediately after birth by a chromogenic substrate assay. Values in umbilical cord blood were identical with values in capillary or peripheral vein blood samples taken within 6 hours after birth. There was no correlation between AT III values and gestational age (r: 0.18). Twenty-four neonates with IRDS showed a mean AT III value of 0.23 U/ml (S. D. ± 0.07 U/ml) which was significantly lower than a mean AT III value of 0.35 U/ml (S. D. ± 0.1 U/ml) for neonates without IRDS (p ≺0.00005). When IVH gr. III/IV was diagnosed in neonates having IRDS (8/24) no significant difference in mean AT IIIact was observed with respect to jnean AT III levels of remaining neonates without this complication. No death occurred in neonates without IRDS. Mean AT IIIact (0.21 U/ml) in neonates with IRDS who died (9/24) was low compared with mean AT III levels of neonates with IRDS who survived (0.25 U/ml), but did not reach significance (p≻0.1). Assuming a critical value of AT III of 20% a positive predictive value of 89% for IRDS, 44% for IVH, and 56% for death was calculated. It is concluded that low AT Illact levels have a high predictive value for IRDS.
PROSTACYCLIN PRODUCTION BY UMBILICAL ARTERIES FROM PREGNANCIES COMPLICATED BY SEVERE PREGNANCY INDUCED HYPERTENSION
Authors: McLaren M , Greer I T A , Forbes C D
Published: June 2019

Approximately ten per cent of allpregnancies are complicated by some degree of hypertensive disease. Since the level of blood pressure is closely related to foetal well-being, if the blood pressure is consistentlyelevated during pregnancy, there is an associated increase in perinatal mortality. Various workers have suggested involvement of prostaglandins in the pathogenesis of pregnancy induced hypertension (PIH). One of our previous studies showed unrecordablelevels of plasma prostacyclin metabolites (PGI2m) of <5pg/ml in all hypertensive patients during the third trimester, whereas none of the normalgroup developed unrecordable levels at any stage.

In this study we used an umbilical artery perfusion model to compare the ability of normal platelet poor plasma, which has been shown to contain a PGI2 stimulating factor, to stimulate production of PGI2 from arteries from normal pregnancies and pregnancies complicated by PIH. Arteries from two patients suffering from PIH with persistent diastolic pressure of >90mm mercury were exhausted of spontaneous PGI2 production beforeperfusion with normal platelet poor plasma. In each case an artery from a normal pregnancy was used as a control. In each experiment levels of PGI2 rose from a baseline level of<50pg/ml to <1000 pg/ml in the normal arteries, whereas the arteries from PIH patients showed no significant rise. It would seem therefore that ourfinding of unrecordable levels of plasma PGI2m in hypertensive pregnancies may be related to defective endothelial cell production.
CONGENITAL HYPOFIBRINOGENEMIA, DEEP VEIN THROMBOSIS AND RECURRENT PLACENTAL ABRUPTION
Authors: Godal H C , Brosstad F , Holm B
Published: June 2019

A patient with hypofibrinogenemia, deep vein thrombosis and recurrent placental abruption is reported. The persistently low plasma fibrinogen concentration strongly suggested that the condition was inherited, and this was supported by low fibrinogen levels and thrombosis tendency in the patient's brother. The exact nature of the condition is not known, but the close agreement between the clottable and immunologic fibrinogen -indicates the presence of a hyperfibrinogenemia sensu stricte rather than dysfibrinogenemia, and this was supported by uniformly negative tests to detect fibrinogen abnormalitied.

Up to now, less than 50 cases of congenital hypofibrinogenemia have been reported. It is not entirely clear whether hypofibrinogenemia represents the heterozygous form of afibrinogenemia or whether itrepresents some sort- of dysfibrinogenemia with abnormal rate of biosynthesis but normal clotting and crosslinking functions. Four of the reported patients with hypofibrinogenemia had placental abruption, a complication which occursin less than of pregnancies. In addition, a relatively high proportion of patients withcongenital hypofibrinogenemiahad increased tendency to thrombosis. On this background it is tempting to speculate that the elevated fibrinogen during pregnancy serves other functions than to facilitate the formation of haemostatic plugs. Thus, during pregnancy and other socalled "acute phase" situations, minute amounts of fibrin may be generatedwithin the circulating blood. Since, moreover, the solvent for these fibrin molecules isfibrinogen, the possibility exists that hyperfibrinogenemia contributes to the prevention of thromboembolism by keeping trace amounts of fibrin in solution.
ANTICOAGULANT AND ANTIGENIC LEVELS OF PROTEIN C AND PROTEIN S IN PATIENTS ON STABILIZED ORAL ANTICOAGULANT TREATMENT
Authors: D'Angelo A , Gilardoni F , Seveso M P , Poli P , Quintavalle R , Manotti C
Published: June 2019

Isolated deficiencies of protein C and protein S, two vitamin K-dependent plasma proteins, constitute about 70% of the congenital abnormalities of blood coagulation observed in patients with recurrent venous thrombosis beLow the age of 40. The laboratory diagnosis of congenital deficiency of these proteins represents a major problem since a large proportion of patients are on oral anticoagulation (OA) at the time the deficiencies are suspected.Under these circumstances the availability of a reference interval obtained in patients on stabilized OA has proven useful.

Functional (C) and antigenic levels (Ag) of protein C, protein S, factor IX and II were estimated in 136 patients on stabilized OA, subdivided according to the degree of anticoagulation (Internatio nal Normalized Ratio, INR).

The results indicate that with increasing anticoagulation the activity levels of all the vitamin K-dependent factors decrease to a greater extent than the corresponding antigenic levels. At variance with the other factors, total protein S antigen levels are only moderately reduced by OA with protein S anticoagulant activi ty comparing well to factor IX clotting activity. These data suggest the possibility of identifying both quantitative and qualita tive deficiencies of protein C and protein S in patients on oral anticoagulant treatment.
PLASMA LEVELS OF PROTEIN S AND C4b-BINDING PROTEIN DURING TREATMENT WITH L-ASPARAGINASE
Authors: Viganò D'Angelo S , Gugliotta L , Gilardoni F , Macagni A , Chetti L , D'Angelo A
Published: June 2019

Protein S (PS), the cofactor of activated protein C (PC), circulates in plasma as free PS (active) and in complex with C4b-binding protein (inactive). We have followed the changes of total PS, free PS, PS activity and C4b-binding protein in 5 adult patients with acute lymphoblastic leukemia treated with 20,000 IU/sqm L-asparaginase (L-ASE) administered three times weekly for two weeks (6 doses) and compared them to the changes of PC antigen and activity, antithrombin III and fibrinogen. The table shows the mean values of these parameters (% of pretreatment values) observed after three doses of L-ASE (A), after completion of treat ment (B) and one week after the end of treatment (C).

These data suggest that at variance with PC and similar to fibri nogen and antithrombin III, total PS antigen is still reduced af ter one week from the end of L-ASE treatment. The reduction of total PS reflects that of free PS, as predicted by mass action. On the contrary, PS anticoagulant activity is decreased to a higher extent than total PS antigen. Acquired PS deficiency might play a contributory role to the development of thrombotic comply cations in patients receiving L-ASE.

Supported in part by CNR grant n 85.02200.44
DEVELOPMENT OF A PROTEIN C CONCENTRATE
Authors: Bhattacharya Prabir , Orthner Carolyn L , Strickland Dudley K
Published: June 2019

A Protein C (PC) concentrate may be useful in treating patients with congenital or acquired Protein C deficiencies. A method for preparation of a human Protein C concentrate has been developed using a by-product of American Red Cross Factor IX production as the starting material (Menache et. al. Blood, 64, 1220). Levels of other vitamin K dependent proteins in the Protein C concentrate were measured and found to be <10 units per 100 units of PC, except for Protein S. The level of Protein S as judged by immunological assay was 30 u/100 u PC. Assay of the PC concentrate using chrcmogenic substrates revealed that levels of thrombin, Factor 3�a and Factor IXa were less than 0.006 u/mL. In addition, Antithrombin III and ax -macroglobulin were not detected. The vivo effects of Protein C concentrate and Protein C activated by thrombin have been tested in anesthetized rabbits. Thrombin was removed from the activated Protein C by ion-exchange chromatography; depletion was verified by S-2238 or by a clotting assay (< 0.006 u/mL). Rabbits were injected with Protein C concentrate (400 ug/kg) or activated Protein C 24 - 48 ug/Kg). The activated partial thromboplastin time (APTT), FactorV (FV) and Factor VIII (FVIII) levels were measured in samples collected over the next three hours. Infusion of PC concentrate elevated the level of PC to 150% of the preinfusion level within 30 min. It did not change the levels of FV, FVIII, fibrinogen or platelet count. In contrast, infusion of activated Protein C produced progressive prolongation of the APTT. Levels of FV and FVIII were decreased to 25% and 50% of preinfusion levels, respectivelv, three hours after the infusion. Fibrinogen and platelet levels were unchanged during that period. These data demonstrate that activated human Protein C concentrate induces an anticoagulant effect that can be readily measured in rabbits.
The Effect of Endotoxins on Generation of Thrombin in the Platelet Atmosphere
Authors: Vermylen J. , Fumarola D. , Semeraro N.
Published: June 2019

Human platelets, washed by repeated albumin density gradient centrifugation, aggregate strongly, occasionally in 2 waves, 3 to 5 minutes after addition of both calcium ions (2.10−3 M f.c.) and one tenth volume normal human serum, provided the serum contains at least 0.6% residual prothrombin. This aggregation is prevented by heparin or hirudin. Samples removed at the onset of aggregation rapidly clot purified fibrinogen, whereas the serum-CaCl2 mixture alone does not clot purified fibrinogen within 24 hours. It is therefore concluded that thrombin is rapidly generated in the platelet atmosphere.

The in vitro effect of endotoxins on human, in contrast to rabbit, platelets is not well established. Using eight different bacterial lipopolysaccharides we failed to demonstrate aggregation in human platelet-rich plasma or of washed platelets or to find significant 14C-serotonin release. On the other hand, all these endotoxins in a final concentration of 100 μg/ml consistently shortened the latent period before aggregation in the washed platelets-serum-CaCl2 system. It is concluded that bacterial endotoxins enhance the generation of thrombin in the atmosphere of human platelets.
Effect of ADP and Thrombin on Fibrin Retraction Inducde by Human Platelets and Fibro-Blasts
Authors: Niewiarowski S. , Stewart G. J. , Goldstein S.
Published: June 2019

Clots formed upon addition of thrombin to suspensions of human platelets (107–109/ml) or of cultured human skin fibroblasts (2 × 106/ml) and fibrinogen (0.1%) retracted within 10–120 min at 37°C., however, when thrombin was substituted with Bothrops marajoensis thrombin like enzyme (BM enzyme) no retraction occurred. ADP (50 η moles) and low concentrations of thrombin (0.01–0.1 units) stimulated platelets to retract clots formed with BM enzyme. This type of clot retraction was inhibited by apyrase and creatine phosphate kinase in the presence of creatine phosphate but that induced by higher concentrations of thrombin (2 units) was not affected. Since thrombin releases ADP from platelets it is possible that clot retraction induced by low concentrations of thrombin is mediated by ADP. On the other hand high concentrations of thrombin may promote clot retraction independently of ADP. As tested by firefly method fibroblasts contained 12 n moles ATP and 4 n moles ADP per mg protein, a value closely ressembling that of nor metabolic adenine nucleotides in platelets. Thrombin did not cause release of adenine nucleotides from fibroblasts nor did addition of ADP stimulate fibroblasts to retract fibrin. These data support the view that the response to ADP is a specific cellular property of platelets and suggest that both types of cells may have similar receptors sensitive to thrombin.

(Aided by grants HL 15226 and HL 14217 from N.I.H. and MA-3515 from the Medical Research Council of Canada.)
Endogenously Increased Fibrinolytic Activity Induced by Liver-Bypass in Pigs and its in Vitro Synergism with Synthetic Fibrinolytic Compounds
Authors: Ostendorf P. , Kaulla K. N. von
Published: June 2019

The marked increase of fibrinolytic activity during the unhepatic phase during liver transplantation in animals and man is well known. Based on these observations the liver-bypass in the pig was used for producing endogenous increase of fibrinolytic activity of several hours duration thus providing fibrinolytic blood specimens suitable for in vitro studies of the synergism of endogenously induced fibrinolytic activity with the fibrinolytic activity induced by synthetic compounds. Liver-bypass in the pig results within a few minutes in a marked drop of the euglobulin lysis time from an average of 16 hrs. to one hour or less. Whereas the antiplasmin activity does not show any clear-cut change during the bypass, the antiactivator activity had dropped after about 2 hrs. to half of the original values. The fibrinolytic activity of the lover-bypass plasma is markedly enhanced by synthetic fibrinolytic compounds such as flufenamic acid applied at concentrations which themselves do not induce fibrinolytic activity. This can be demonstrated in various ways such as the use of thrombelastograms or lysis of clots formed by the bypass plasma. Also, as the bypass goes on, the clots start to dissolve spontaneously and the lysis time is considerably shortened by addition of synthetic compounds. Seen from the point of view of a potential clinical use, the most pertinent observation is that plasma obtained during an early bypass phase which does not dissolve preformed human clots, does so after addition of synthetic compounds.
Functional and Metabolic Studies of Human Asialofibrinogen
Authors: Martinez J. , Palascak J. E. , Peters C. L.
Published: June 2019

Human fibrinogen treated with vibrio cholerae neuraminidase released over 90% of its sialic acid, as measured by the thiobarbiturate method, without any alteration in peptide chain structure on SDS-acrylamide gel electrophoresis. The thrombin time of asíalo -fibrinogen was significantly shortened when compared with normal fibrinogen. To study the ability of human asialofibrinogen to cross-link under the influence of Factor XIII, normal fibrinogen and asialofibrinogen were each clotted with thrombin in the presence of Factor XIII and calcium. At time intervals ranging from 3 minutes to 2 hours clots were removed, reduced with mercaptoethanol, solubilized and run in SDS-polyacrylamide gels. Normal and asialofibrin show similar rates of γ–γ chain dimerization, followed by α-chain polymerization that is complete within 2 hours of incubation. In contrast to previous studies, our data show that asialofibrinogen has a normal ability to form cross-linked fibrin. The metabolic properties of human asialofibrinogen were investigated by simultaneous infusion of 131I-normal fibrinogen and 125I-asialofibrinogen into rabbits. In contrast to most asialoproteins, the half-life of asialofibrinogen was decreased by only 26% when compared with 131I-normal fibrinogen. Thus it appears that sialic acid is neither a major determinant in fibrinogen catabolism nor in normal cross-linking of fibrin.
γ’-Dimer Antibody and its Specificity Against Fibrinogen, FR-a and Carboxymethylated chains from Fibrinogen and Cross-linked Fibrin
Authors: Clausen N. T. , Gormsen J.
Published: June 2019

γ’-dimers (M: 80.000) were obtained from fully cross-linked fibrin (Kabi, grade L, 96% clottable) by digestion with porcine plasmin (0.5 CTA units/mg protein) for 18 h., carboxymethylation and subsequent isolation on Whatman CM-52 with a combined pH (4.8 → 5.5) and ionic strength (0.05 → 0.25 M) gradient in Na-acetate buffer with 8 M urea*).

The γ’-dimers were recycled once and exhibited purity on SDS polyacrylamide gel electrophoresis. These γ’-dimers showed weak reaction in immunoprecipitation assays with anti-fibrinogen and anti-D but not with anti-E rabbit immunserum.

Heterologous antibody was obtained from rabbit after subcutaneous injections of γ’-dimer antigen and bleeding after 4 months, 14 days after last injection.

This crude antibody reacted in immunodiffusion and monorocket immunoelectrophoresis with γ’-dimers and weakly with fibrinogen and fragment D but not with fragment E (DEAE purified). After absorption with fibrinogen and fractional desalting with (NH4)2SO4 (25% and 50%) there was no reaction with fibrinogen, D or E fragments, but still with γ’-dimers.

Testing against isolated chains (SDS-pure) from fibrinogen revealed Aα cross-reaction but only reaction with γ and γ’-dimer chains remained after proper absorption. Possible differentiation between these two subunit structure is reported.

* Murano, G. et al. FEBS-letters 14, 37–41, 1971.
In Yivo Behavior of Human Fibrinogen and Fragments X, D and E.
Authors: Ardaillou Nicole , Dray Lise , Larrieu Marie-José
Published: June 2019

Fragments X, D and E were obtained from plasmin digestion of purified human fibrinogen (fraction I-O). Fragment × was isolated from stage 1 digest by filtration on Sephadex G-200 by Budzinski and Marder, and fragments D and E in our laboratory by Pevikon block electrophoresis. The purified fragments were identified by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. Human fibrinogen and fragments X, D and E were labelled with 125iodine by the lactoperoxidase method. Plasma disappearance curves were studied in rabbits. A two exponential curve was obtained with all of them, with second exponential (lower slope) being clearly predominant. The half-lifes of the second exponentials were different for each fragment: there appears to be a relationship between the molecular weight of the fragments and their half-lifes. Values observed (mean±s.e.m.) were: fibrinogen 49.34±5.4 hours, fragment X 6.14±0.44 hours, fragment D 2.25±0.20 hours and fragment E 1.53±0.13 hours. No fragment was found either to polymerize or to bind to plasma proteins in vivo following injection into rabbits as demonstrated by the elution peaks obtained after filtration through Sephadex G-200.
Approaches to the Structure of Fibrinogen
Authors: Finlayson J. S.
Published: June 2019

The history of the struggle to comprehend fibrinogen is written in individual experiments of individual investigators. Nonetheless, much of the strategy used in approaching its structure can be classified into a few overlapping categories. One class comprises chemical and immunological analyses. The former have yielded considerable progress toward the primary structure and have revealed various types of heterogeneity. The latter have demonstrated both homologies and domains, and may prove invaluable in phylogenetic and biosynthetic studies ; however, by definition, they are at the mercy of the antibody-producing organism. A second strategic class is model building. Early models were based on data from physical (largely hydrodynamic) measurements ; later ones, on morphological observations. More recent efforts have involved dissective approaches, e.g., proteolytic fragmentation. Though models constructed to date are mutually exclusive in detail, their common structural features and the design of experiments used to test them have not yet permitted unequivocal conclusions. Studies of afibrinogenemia, like organ extirpation, can indicate physiological roles but provide little structural information. By contrast, investigations of congenital dysfibrinogenemia are more analogous to perturbation experiments. In addition to providing well defined examples of permissible structural variations, these investigations carry the potential for revealing important structure-function relationships.
Structural Abnormality in the Gamma Polypeptide Chain of Paris I Fibrinogen
Authors: Budzynski Andrei Z. , Marder Victor J. , Ménaché Doris , Guillin Marie-Claude
Published: June 2019

Congenital abnormal fibrinogen Paris I has an abnormal γ chain which is probably the cause of impaired polyperization of fibrin monomers and an inability to crosslink in the presence of Factor XIII. SDS-polyacrylamide gel electrophoresis demonstrated that the Aα and Bβ chains of Paris I fibrinogen are normal. However, only 1/3 of the γ chains are normal, the remaining 2/3 migrating as a heavier species between the B β and γ chains. The conversion of Paris I fibrinogen into crosslinked fibrin in the presence of thrombin, calcium and Factor XIII is accompanied by the disappearance of the normal γ chain and the formation of γ chain dimers, but the abnormal γ Paris I chain did not disappear. There was only partial disappearance of the α chains, with approximately 2/3 not participating in the crosslinking reaction. It would appear that 2/3 of the fibrinogen molecules in the plasma of the propositus have abnormal γ Paris I chains, which are approximately 2500 daltons heavier than the normal γ chains. Plasmic digestion of Paris I fibrinogen results in the formation of an abnormal Fragment D with a heavier γ chain remnant. Degradation of Paris I fibrin yields a normal Fragment D-D and an abnormal Fragment D. The data indicate that the structural defect in γ Paris I chain occurs in the carboxy-terminal region, which can account for both the defective polymerization and imperfect crosslinking.
Recovery of the Venous Wall from Leukocyte Induced Injury
Authors: Stewart G. J. , Ritchie W. G. M. , Lynch P. R.
Published: June 2019

Surgical trauma to tissue adjacent to canine jugular veins combined with brief local stasis caused massive leukocyte invasion of venous walls. This resulted in the entrapment of pockets of leukocytes between the endothelium and basement membrane and subsequent detachment of patches of endothelium.

By an hour smooth muscle showed a decrease in myofibrils and an increase in rough endoplasmic reticulum and collagen lost its ability to stain with uranyl acetate-lead citrate.

By 24 hours smooth muscle cells had undergone various changes and formed a partial covering layer over the surface of denuded areas, often by extending long “arms” from a bulky, fibroblast-like “body”. Various stages of de-differentiation and re-differentiation were abundant. By 72 hours these cells had become thinner and resembled the “immature” endothelium of Florey and others. However, the sheet was still discontinuous. By 7 days and thereafter to 28 days the endothelium was continuous and typical of “immature” endothelium morphologically but the surface membrane stained intensely in some instances.

At 24 hours there was a perivascular zone of edema with cell ghosts and amorphous debris which decreased thereafter. Collagen staining was again typical by 7 days.

These observations indicate that the luminal surface of blood veins can be initially repaired by the rapid de-differentiation and re-differentiation of smooth muscle rather than waiting for the ingrowth of endothelial cells from the margins of the denuded areas.

(Supported by N. I. H. Grants ≠ HL 14217 and HL 08886.)
Binding of Collagen to Platelets
Authors: Jaffe R. M. , Deykin D.
Published: June 2019

Type I (chick calvaria) and Type II (chick cartilage) collagen were synthesized in organ culture on tritiated amino acids. The final, purified collagen has sp. act. > 106 DPM/mg. protein.

3H-Tropocollagen, from 5000–40,000 molecules per platelet, does not bind during a 15 minute observation period. Multimeric collagen, at the same dose, binds to platelets after a lag period of 90–120 sec. Fibrillar collagen will bind within 10 sec. Gelatin, prepared from this radiolabeled collagen, does not bind during the observation period. Similar results obtain for both Type I and Type II collagen.

Aggregation and binding studies were carried out simultaneously. Tropocollagen does not initiate aggregation; multimeric collagen initiates aggregation after a lag of about 180 seconds; fibrillar collagen initiates aggregation with a lag of about 60 seconds. Gelatin does not initiate aggregation. Thus binding precedes aggregation by about 60 seconds. These observations are consistent with a quaternary requirement for platelet aggregation induced by collagen.

Chemical modification of ॉ-amino groups of lysine residues in collagen was performed. Certain modifications inhibit fibrillogenesis and/or denature the protein. These modifications inhibit collagen-induced platelet aggregation. Permethylation of lysines in collagen does not impair fibrillogenesis, does not denature the protein and does not inhibit collagen-induced platelet aggregation.
Quantitative Determination of Collagen-platelet Binding
Authors: Kronick P. L. , Jimenez S. A.
Published: June 2019

We have investigated the structural features of the collagen molecule necessary for adhesion to platelets. The use of biosynthesized14–proline labelled collagens allowed us to measure directly and quantitatively the very small amounts of chicktendon and cartilage collagen protomers which bind to platelets and compare their binding with that of the corresponding fibrils. The binding of fibrillar aggregates separated by centrifugation was found to increase with sedimentation coefficient up to a limit of s = 15-S. The fraction of 15-S aggregates bound was at least four fold greater than that of 5-S aggregates. The highly glycosylated cartilage collagen allowed to aggregate at 4° C by prolonged storage yielded a product 50% of which bound to platelets ; fresh cartilage collagen and procollagen bound less than 5% both at 4° C and 37° C. Lowering the pH from 7.4 to 6.5 had no effect on the binding of the aggregates.

The quantitative binding parameters which we have determined show that collagen must be in an aggregated form to bind to platelets ; the extent of glycosylation does not exert a great effect on the binding tendency and the amount of collagen in protomeric form which can bind to a platelet under physiologic conditions is very small.

(Supported by NIH Contract No. 1-HB-42982.)
Collagen Coated Gelatine Tubes for the Investigation of Platelet Adhesion and Subsequent Platelet Aggregation Under Controlled flow Conditions
Authors: Muggli R. , Baumgartner H. R.
Published: June 2019

Aggregometer studies do not discriminate between platelet adhesion and platelet aggregation. Therefore, we prepared a homogenous collagen surface which could be exposed to whole blood in a perfusion chamber under controlled flow conditions.

Artificial “vessel” segments were prepared by dipping glass rods into 20% gelatine and, after air-drying, cross-linking the gelatine in 2.5% glutaraldehyde. Segments of 1 cm length were then drawn on the rod of the perfusion chamber and coated with 300 μl of neutral salt soluble collagen (2.2 mg/ml). Surface coverage with collagen was virtually complete (96%–100%).

Uncoated or collagen-coated gelatine segments were exposed to citrated rabbit blood for periods up to 40 min. Platelet-surface interaction was evaluated morphometrically. On uncoated segments surface coverage with platelets amounted to 31% and 50% after 10 min and 40 min (the corresponding ratios of contact/spread platelets were 2.6 and 1.5). Only 0.1% thrombi were found. On collagen-coated segments surface coverage with platelets amounted to 57% and 83% after 10 min and 40 min (the corresponding ratios of contact/ spread platelets were 0.1 and 0.0); platelet thrombi were found on 33% and 42% of the surface after 10 min and 40 min.

Platelet adhesion and subsequent aggregation on the collagen-coated artificial surface is similar to that observed on α-chymotrypsin digested subendothelium. The results suggest that fibrillar collagen triggers rapid spreading on a surface, a reaction which is closely associated with the formation of platelet thrombi. The latter phenomenon is thought to be caused by the release of aggregating agents from the spreading platelets.
Platelet Adhesion to Insolubilized Aggregating Agents
Authors: Heene D. L. , Grotemeyer G. , Matthias F. R. , Lasch H. G.
Published: June 2019

Platelet aggregating substances, such as bovine fibrinogen (FG) and collagen (Col), were insolubilized by means of chemical fixation to CNBr-activated agarose. Platelets exposed to bovine FG-ag or Col-ag readily adhere and “aggregate” to these insolubilized agents. An assay system was designed which indicates the platelet reactivity towards FG-ag and Col-ag by means of the decrease of platelet count in platelet rich plasma after exposure to the agarose bound aggregating substance. After evaluation of the standard conditions concerning reaction time, amount of substrate and degree of disappearance of platelets for PRP from healthy donors (arbitrarily set to 100% ) the procedure was applied to clinical cases and revealed significant (p < 0.001) increase of platelet reactivity in patients with venous thrombosis (n = 11), arterial occlusive disease (n = 12), hyperlipemia (n = 26) and storke (n = 6). Mainly the FG-ag test was found to be suitable to detect hyperactivity (up to 300%) in cases with thromboembolic complications and to control the effect of ASA.
Effect of Methotrexate and Vincristine on rat Bone marrow Megakaryocytes and Platelet Function
Authors: Jerushalmy Z. , Pinkhas J. , Krinsky M. , Vries A. de
Published: June 2019

Methotrexate administered intraperitoneally to rats caused a transient disturbance in platelet function manifested by absence of ADP-induced aggregation and serotonin incorporation, and by decreased factor 3 (PF3) availability. These phenomena were noticed when the megakaryocyte count decreased while the platelet count was still within the normal range.

Vincristine injection was not followed by an immediate drop in megakaryocyte and platelet count or PF3 availability, however a transient decrease of ADP-induced platelet aggregation and serotonin incorporation occurred.