bluuebunny/crossref_metadata_2025 huggingface dataset:
VERY LARGE AND COMPACT AGGREGATES OF RED CELLS IN HEART DISEASE AND CANCER: POSSIBLY AN ANALOGOUS ROLE IN THE MICROCIRCULATION AS PLATELET AGGREGATES
Authors: Dintenfass L
Published: June 2019

Very large aggregates of red blood cells, showing compact morphology, are easily observed in vitro, and might be of importance in the in vivo microcirculation. Blood from patients with myocardial infarction, WaldenstrBm1s macroglobulinaemia, or varoous carcinomas. etc., was anticoagulated with EDTA, and adjusted to haematocrit of 0.30, using native plasma. All tests were carried out in the slit-capillary photoviscometer, at temp, of 22°C. Micro and macrophotographs were taken during flow and stasis, using slits of 12.5 and 50 micron gaps. Studies showed that very large (two-dimensional) red cell aggregates are formed, such aggregates (clumps) containing up to 50,000 red cells in a single clump. The architecture of such aggregates differed according to the origin of blood: both rouleaux type and random / compact type of aggregates were observed; in principle, a spectrum of morphologies can be seen. These observations form a link between the earlier work of FAHRAEUS, on the one hand, and that of KNISELY, on the other; whic works appeared at the time to be contradictory and irreconcilable.
INTERACTION BETWEEN LUPUS ANTICOAGULANT AND PLATELETS
Authors: Corte-lazzo D , Galli M , Viero P , Barbui T
Published: June 2019

Lupus Anticoagulants (LAC) are IgG or IgM immunoglobulins which interfere with phospholipid-dependent coagulation tests and actively react also with platelet wall phospholipids. This interaction may result in platelet quantitative and qualitative defects. We have examined 10 patients with LAC diagnosed on the basis of the commonly accepted criteria (Working Party reccomandations, 1983). Four had concomitant Systemis Lupus Erythematosus, one Waldenstrom's disease (W. D.) and five no apparently underlying disease. Only the case with W. D. presented bleeding tendency, whereas the others had a history of thrombotic complications. A vei?y striking defect of platelet aggregation by ADP, epinephrine, collagen and arachidonic acid was documented in the case of W. D., who had also a very prolonged bleeding time (>20 minutes) ; ,in this latter case LAC was an IgM. In the others, LAC, identified as IgG immunoglobulins, produced no aggregation abnormalities. On the contrary, serotonin platelet concentration, platelet, plasma and urine Beta-Thromboglobulin contents showed values consistent with a pattern of platelet activation in all cases. IgG immunoglobulins separated from sera of six patients showed LAC activity but no effects on platelet aggregation of a normal PRP, whereas induced secretion of Beta-Thromboglobulin from normal platelets.
A STANDARD ANTI-CARDIOLIPIN (aCL) TEST: USEFULNESS IN IDENTIFICATION OF THE ANTI-PHOSPHOLIPID SYNDROME
Authors: Harris EN , Asherson RA , Baguley E , Ridley M , Hughes GRV
Published: June 2019

Some, but not all, patients with the lupus anticoagulant and anti-cardiolipin antibodies are prone to thrombosis, fetal loss and thrombocytopenia. It will be important to identify the particular sub-group of patients with anti-phospholipid (aPL) antibodies most subject to these clinical disorders. A preliminary study has shown that the level of IgG aCL antibody is predicitive for thrombosis, fetal loss, and thrombocytopenia but it will be difficult to substantiate (or refute) these findings unless there is a uniform system to measure aCL antibody levels.

Five test sera with defined IgG and IgM aCL levels are currently available to laboratories wishing to standardise the aCL test. The concentrations of aCL in these sera cover the full sensitive range of aCL solid phase assays. Using these. 5 test sera to calibrate our assay system, sera from 3000 patients were analysed: 1400 healthy adults and 1600 consecutive patients with autoimmune disorders. All sera from healthy adults had aCL levels below 10GPL (IgG aCL) or below 10MPL (IgM aCL) units. Of the 1600 autoimmune patients, 115 had levels above 5 GPL and/ or 5MPL units. More than 2/3 of patients with IgG aCL levels above 20 GPL units (30 patients) had thrombosis or fetal loss, but the frequency of these disorders decreased in the 10-20 GPL and 5-10 GPL unit groups. Of the 9 patients with IgM aCL levels above 15 MPL units, 6 had thrombosis or fetal loss.

The availability of reference sera to measure IgG and IgM aCL antibody levels may better enable multicenter studies to be performed. A relatively uniform system of measurement may also enable easier identification and management of patients with the anti-phospholipid syndrome.
PROTEIN S AND ANTICARDIOLIPIN ANTIBODIES IN PATIENTS WITH LUPUS ANTICOAGULANT WITH OR WITHOUT THROMBOSIS
Authors: Panzer S , Pabinger I , Dudczak R , Schulz E , Wurz E , Lechner K , Lechner K
Published: June 2019

Arterial and venous thrombosis frequently occur in patients with lupus anticoagulant. We investigated in patients with lupus anticoagulant the possible association between protein S:Ag deficiency and/or anticardiolipin antibodies and a history of thrombosis. In 27 patients (8 with and 19 without a history of thromboembolic disease) free protein S:Ag was determined with immunoelectrophoresis in PEG 8000 precipitated plasma. Anticardiolipin antibodies were measured in 22 patients (10 with thromboembolism) by means of a solid phase radioimmunoassay.

Free plasma protein S:Ag was normal or elevated in 25 patients and slightly decreased (protein S:Ag 67%) in 2 patients without thromboembolism. 2-dimensional electrophoresis revealed a normal distribution of the free and of the complexed form of protein S:Ag. Anticardiolipin antibodies were found in 6 out of 10 patients with a history of thrombosis (5 of 7 with venous thrombosis, 1 of 1 with cerebral infarction , none of 2 with combined venous and arterial thromboembolism) and in 4 out of 12 patients without a history of thrombosis.

We conclude 1. the thrombophilic state in patients with lupus anticoagulant cannot be explained by a reduction of protein S:Ag, and 2. there seems to be no correlation between the presence or absence of anticardiolipin antibodies and the development of thromboembolic disease.
RECOMBINANT ALPHA-1-ANTITRYPSIN PITTSBURGH ATTENUATES EXPERIMENTAL GRAM-NEGATIVE SEPTICEMIA
Authors: de La Cadena R A , Flores D , Scott C F , Cousens L , Barr P J , Hoffman I B , Kueppers F , Fisher D , Pissarello G , Colman R W
Published: June 2019

Gram-negative septicemia is known to be associated with activation of the contact system and disseminated intravascular coagulation. Alpha-l-antitryosin-Pittsburgh (AT-P), a naturally occurring lethal nutation (358Met →> Arg) is a potent inhibitor of all the contact enzymes as well as the coagulation enzyme, thrombin. We hypothesized that the administration of AT-P might modulate the course of gram-negative sepsis. The pig was selected as the animal model of choice since its fibrinolytic, kinin-forming, and coagulation systems are similar to man as well as its response to endotoxin. Young pigs were infused with viable pseudomona aeruginosa (2x108 CFU), and were either untreated (Group I) or treated (Group II) for 6h, with recombinant AT-P (rAT-P), produced in yeast. Coagulation and hemodynamic parameters as well as survival rates were evaluated. The mean pulmonary artery pressure, mean systemic blood pressure, white blood count and platelet count were the same in both groups during the entire experiment. rAT-P was removed by immunoadsorp-tion prior to assay of coagulation parameters. At lh, the changes for all coagulation descriptors, AT-I II, Factor XI, fibrinogen, and fibrinogen degradation products were statistically significant (pπ.05). Prekal1ikrein levels, however, were not statistically different between groups. Survival rates in Group I were significantly less than Group II (pπ.05), during hours 2-5, but did not differ at 6h, when the functional plasma rAT-P levels were lowest. A discrepancy was observed between antigenic and functional levels of rAT-P that increased linearly during the course of the experiment. This suggested that activation of proteases was occurring with subsequent inactivation by rAT-P. This study indicates that rAT-P affords protection in experimental gram-negative septicemia by modulation of the activity of the serine proteases resulting from activation of the contact phase of blood coagulation, fibrinolysis, kallikrein and thrombin formation.
THE EFFECT OF APROTININ ON EXPERIMENTAL PORCINE SEPTIC SHOCK
Authors: Svartholm E , Haglund U , Ljungberg J , Hedner U
Published: June 2019

Septic shock induces pronounced haemodynamic changes resulting in pulmonary microemboliz., complement activ. and an activ. of the coag. and fibrinol. systems. Only minor effects on the sepsis-induced changes were found on pretreatment with a variety of platelet aggr. inhib. (Svartholm et al 1986). Aprotinin inhibits among other enzymes kallikrein. Endotoxin shock was induced in 12 pigs by i .v. inf. of E.coli, six of vdiich got Aprotinin i.v. (1000000 KIE) immediately before the E.coli infusion. Another 6 pigs were used as controls. Haemodynamic parameters, platelet counts, fibrinol. act., fibrinogen, ATIII, and ethanol gel. test were measured before the infusions, 8,60, 90-120, 150-180 min after. Ihe haemodynamic parameters stayed unchanged in the control pigs. In the E. coli-treated animals cardiac output and the mean art. pressure decreased signif. On the other hand, the mean pulm. art. blood pressure and the pulm. vase, resist, increased signif. Ihe increase of the pulm. resist, was less pronounced and reversible in the Aprotinin treated animals. Ihe pulm. vase, resist, in the E.coli group increased contin. from 2.8ଐ.2 mm Hg before to a max. of 23.4 mm Hg at the end of the obs. time and in the Apro-tinin-treated animals from 2.0ଐ.3 to 6.6଑.9 mm Hg (8 min after the inj.) being down to 4.4ଐ.2 at the end of theexp.

The platelet decrease was less pronounced in the Aprotinin pretreated group, the ethanol gel test turned pos. in the E.coli group after 60 min but stayed neg. in both the contr. and the Aprotinin pretreated animals. Fibrinolytic act, was seen in all E. coli-treated animals but stayed neg. in the contr. Moderate changes were seen in ATIII and fib. gen in all E.coli-treated pigs. All animals treated with only E.coli died at the end of the obs. time but none in the control group and none in the Aprotinin pretreated group

In conclusion Aprotinin seems to prevent the secondary fatal increase in pulm. resist, most probably caused by pulm. oedema and a gen. activ. of the coag. syst. (ethanol gel. test stayed neg.)
FACTOR VIII AND FACTOR XII LEVELS IN BORDERLINE HYPERTENSION
Authors: Patrassi G M , Santarossa A , Fallo F , Sartori M T , Viero M , Girolami A
Published: June 2019

Borderline hypertension causes mortality and morbidity rates similar to those associated with estabilished hypertension. However, there is no univocal guideline for its therapeutic management. Hypercoagulability in hypertension has been demonstrated. The aim of our study was to evaluate some coagulation factors in agroup of patients affected by borderlinehypertension. The following tests were carried out: PT and PTT, Factor VIII coagulant activity, FVIII antigen and FVIII ristocetin cofactor, Factor XII and Factor XI activities. These tests were selected for their relationship to the contact coagulative activation near the vascular wall. In our patients statistically significant higher FVIII and FXII coagulant activities than normal control subjects were found. Moreover, an evident even though not statistically significant PTT shortening was seen. Other tests taken into consideration were all within normal limits. Our results suggest that an increased FVIII and FXII synthesis and/or release is present, and an activated coagulation system exists in borderline hypertension. Furthermore, it is not clear why an excess of FVIII:C over FVIIIR:Ag and FVIIIR:RCof was found in our patients. In conclusion, an activation of haemostatic mechanism was found in borderline hypertension. The young age of patients and the absence of evident hypertensive angiopathy are in agreement with an overactivity of blood vessel tone. Haemostatic activation could be an useful marker in favour of the precious management of patients with borderline hypertension.
TREATMENT OF RAISED BLOOD PRESSURE WITH NISOLDIPINE REDUCES "SPONTANEOUS" PLATELET AGGREGATION IN WHOLE BLOOD
Authors: Wilson J , Orchard M A , Spencer A A , Davies J A , Prentice C R M
Published: June 2019

Hypertensive patients are at risk of premature vascular disease, and abnormalplatelet function in hypertensive subjects may contribute to vascular damage. Ina placebo controlled, double blind, cross over study, we have investigated theeffect of treatment of moderate hypertension with nisoldipine (a 1,4, dihydropyridine calcium antagonist) on several aspects of platelet function. In 12 hypertensive subjects, venous blood samples were taken for platelet count, PCV, βTG, PF4, and aggregation to standard doses of ADP and adrenaline in whole blood. Platelet aggregation in whole blood which occurred during processing (spontaneous aggregation) was also recorded. Samples were collected on four occasions: after 4 weeks treatment with placebo (A), after 6 weeks and 12 weeks of the crossover phase with either nisoldipine (B) or placebo (C), and finally after re-establishment of blood pressure control on routine therapy (D). Nisoldipine was effective in lowering blood pressure, with mean values during the 4 treatment phases of: A 119 mmHg, B 104 mmHg (p < 0.01), C 114 mnHg, D 103 mmHg. Neither nisoldipine nor routine treatment significantly affected platelet count, PCV, βTG, PF4 or aggregation in whole blood to adrenalin or ADP. "Spontaneous aggregation" in whole blood however, was significantly inhibited by reduction in blood pressure during treatment both with nisoldipine and routine drugs. Treatment of hypertension appears to raise the threshold of blood platelets for aggregation regardless of the pharmacological agent used.
LOW PLASMA CONCENTRATIONS OF C4b-BINDING PROTEIN AND VITAMIN K-DEPENDENT PROTEIN S IN PRETERM INFANTS WITH DECREASED FORMATION OF PROTEIN S-C4b-BINDING PROTEIN COMPLEXES
Authors: Malm J , Bennhagen R , Holmberg L , Dahlbäck B
Published: June 2019

Protein S is a vitamin K-dependent plasmaprotein functioning as a non-enzymatic cofactor to the activated form of protein C in the degradation of coagulationfactors Va and VIIa. In the circulation approximately 60% of protein S is complexed to the complement protein C4b-binding protein (C4BP). Only the remaining, free fraction exhibits protein Ca cofactor activity.

The plasma concentrations of protein S and C4BP were determined in 25 term and 26 preterm infants. Both proteins werequantified with radioimmunoassays. The free, functionally active form of proteinS and the total protein S concentration were determined separately. The level ofC4BP in preterm infants was found to be very low (mean 6% of the adult level). In term infants the level had increased to a mean of 18%. Also the total concentration of protein S was decreased in preterm as well as in term infants; 18% and 31% of the adult level, respectively. Free protein S was the predominant form in plasma representing 83 % of total protein S in preterm and 68 % in term infants. This was probably due to the very low C4BP levels. In adult controls the corresponding value was 34%. The plasma concentration of free protein S in preterm and term infants, when compared to the adult level, was 44% and 66%, respectively. These results demonstrate that although the total protein S concentration in preterm and term infants was very low when compared to adult levels, the difference in the concentration of free, functionally active protein S between infants and adults was less pronounced.
LEVELS AND DISTRIBUTION OF FREE AND C4b-BP-B0UND-PR0TEIN S IN HUMAN FETUSES AND FULL-TERM NEWBORNS
Authors: MOALIC P , GRUEL Y , FOLOPPE P , DELAHOUSSE B , BODY G , LEROY J
Published: June 2019

Levels and plasmatic distribution of protein S were studied on umbilical cordplasmas from 25 normal full-term newborns (N) and 15 normal fetuses (F) between20 and 30 weeks of gestation.
Samples from fetuses were collected for antenatal diagnosis by direct puncture of the umbilical vein under high resolution real-time ultrasound. Total protein S(PS) level was determined using Laurell rocket immuno-electrophoresis (Diagnostica Stago, Asnifcres-France). Free PS wasmeasured using this latter method, afterprecipitation of C4b-BP-bound-PS by polyethylene glycol (PEG). Normal pool plasma, treated as well, was considered as the reference curve. C4b-binding protein (C4b-BP) determinations were conducted by Laurell rocket immunoelectrophoresis. The qualitative distribution of free PS and C4b-BP-bound-PS in plasma was also assessed by crossed-immunoelectrophoresis(CIE). Results (mean - SD) were expressed in percentage, in relation to healthy adults values (n = 15). Low levels of total PS were obtained in all fetuses (16.4 ±4.2) and newborns (36.4 ±9.5) as compared to adults (91.6 ± 12.2). Free protein S level was also decreased both in fetuses (22.2 ±6.0) and newborns (48.5 ± 12.1 versus 89.4 ± 26.3 in adults). At these stages of development, the ratio Free PS / Total PS (both values were obtained according to a reference curve performed with a normal adult pool plasma untreated by PEG) was significantly higher as compared to normal adults (0.82 ±0.07 in F, 0.64 ±0.17 in N and 0.39 ±0.11 in A, p‹0.001, Student t test). The predominance of
free PS was also visualized in the CIE patterns. These data may be explained by undetectable C4b-BP in 21-week old fetuses (‹2% in 10 cases). After the 26th week of gestation C4b-BP level was 7.8 ±7.4 ‹n=5) and reached a value of 19.2 ±15.6 in newborns (adults = 95.7 ±14.7). In human fetus and newborn, PS essentially circulates under free form and this might compensate the decrease of the total PS level.
ANTENATAL DIAGNOSIS OF THROMBOPATHY
Authors: Wautier J L , Gruel Y , Boizard B , Caen J P , Daffos F , Forestier F
Published: June 2019

We previously determined platelet antigens and glycoproteins in the human fetus after 19 weeks of intrauterine life (Blood 68, 488-92,1986). These results obtained in fetuses with normal platelets allowed us to do the first attempt of antenatal diagnosis in Glanzmann thrombasthenia. The fetal propositus was tested with monoclonal (AP2, AP3) or polyclonalantibodies (IgGL) directed against GPIIbllla or platelets antigen (PLA1, Leka). The foetus was found to be heterozygous for GT and similar results were foundafter his birth.

Grey platelet syndrome is a rare congenital platelet defect caracterized by an alpha granule deficiency and is transmitted on the dominant feature. To be able to detect this abnormality before birth we have measured the platelet content of alpha granules.

The amount of Beta thromboglobulin (gTG) at 18 weeks of intrauterine life was32±4.3 mg/109 platelets in normal platelets (adults 60 mg/10^ platelets). The foetus of the mother with grey platelet syndrome was sampled at 19 weeks when the mother was under platelet transfusion and the platelets were studied by electron microscopy and for their BTG content. The platelet morphologyshowed the presence of alpha granules and the $TG content was in the range of the control fetuses (42mg/109 platelets). The baby was born after artificial delivery under platelet transfusion. These results showed that the antenatal diagnosis of thrombopathies is feasible and can permit a therapy to avoid dramatic haemorrhage during pregnancy or delivery.
PROTEIN C AND PROTEIN S IN MILD AND MODERATE PREECLAMPSIA
Authors: Hopmeier P , Halbmayer M , Schwarz H P , Heuss F , Fischer M
Published: June 2019

In normal pregnancy, total protein S antigen and activity have been reported to be markedly reduced, whereas protein C level was found unaltered. In contrast, in severe preeclampsia protein C antigen was found to be considerably reduced. The presentstudy was done to clarify whether similar changes in protein Cwould alsobe observed for the mildand moderatepreeclamptic state andwhether there would be any effects on the level ofprotein S, since nodata on this cofactor in preeclampsia have been reported to date. 4-0 women in the 3rd trimester of pregnancy - 20 with uncomplicated pregnancies and 20 who had developed a mild (n = 14-) or moderate (n = 6) preeclamptic condition - were included in the study. All groups were well matched in age and gestational age. In addition, 20 healthynon-pregnant women served as controls. All probands had normal liver (SGOT, SGPT) and kidney (BUN, creatinine) values and no other medication than oral vitamins was used. Classification of preeclampsia was done according to a modification of the gestosis index of Goecke using an 11 gradeindex system (0 - 11). ProteinC antigen was measured by an enzyme-linkedimmunosorbent assay and protein S by the Laurell rocket technique.For statistics, the Wilcoxon rank sum test was applied

We conclude that in comparison tonormal pregnancies, protein S is found elevated at least in the moderate, and protein C in the moderateas well as in the mild preeclamptic state
MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN
Authors: Hessing Martin , Meijers Joost C M , van Mourik Jan A , Bouma Bonno N
Published: June 2019

Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.
REDUCED PROTEIN S ANTICOAGULANT ACTIVITY IN ESSENTIAL MIXED CRYOGLOBULINEMIA
Authors: D'Angelo A , Gilardoni F , Toschi V , Ciminiello C , Sinico E A , Viganò D'Angelo S
Published: June 2019

Protein S (PS) is found in two forms in plasma, as free PS, which functions as a cofactor for activated protein C, and in e-quimolar complex with C4b-binding protein (C4b-bp), an inhibitor of the complement system. The Kd of the PS-C4b-bp interaction is one order of magnitude lower than the plasma concentration of the two proteins; thus 55-60% of total PS circulates in the bound form. Evidence has been provided that in vitro complement activation does not affect the equilibrium between PS and C4b-bp; however in patients with systemic lupus erythematosus and low C4 levels, a shift from free to bound PS has been observed. To further evaluate the relationship between complement activation and PS distribution we have measured PS and C4b-bp levels in 21 patients with essential mixed cryoglobulinemia (EMC), an autoimmune disorder characterized by cryo-precitable circulating immunocomplexes and associated with vasculitis and thrombotic episodes. EMC patients had cryocrit rangin from 1 to 66% and greatly reduced complement components (Clq: 45%, C3: 71%, C4: 15% of normal). Mean PS activity was significantly reduced inpatients as compared to the control population consisting of 20 age-and sex-matched blood donors (69%, p< 0.001). Free PS was similar in patients and controls, but total PS was lower in EMC patients (82%, p<0.05). Seven EMC patients had C4b-bp levels be low 60%. Thus, reduction of PS activity in patients with EMC is not due to reduced free PS. Cultured endothelial cells synthesize and release PS with reduced specif ic activity. In EMC patients very high levels of von Willebrand factor (313%, p< 0.001) a protein released from endothelial cells, but not of ceruplasmin, another acute phase reactant protein, were observed.In vivo release of PS from en dothelial cells might contribute to reduced PS specific activity in EMC.
CONGENITAL SEVERE PROTEIN C DEFICIENCY IN ADULTS
Authors: Kakkar S , Melissari E , Kakkar V V
Published: June 2019

We (Melissari et al, 1985, T.R. 29 [1985] 641) were the first to identify the occurrence of severe protein C deficiency in an adult with thrombophilia and undetectable protein C levels. This report documents our clinical and laboratory resuts of this patient and his family, as well as another 8 patients, in two more, unrelated families. In these unique families with members suffering from severe protein C deficiency (≤6%), no one had experienced neonatal purpura fulminans. Symptoms started mainly in their early twenties, except in 2 patients who first had symptoms at the ages of 11 and 13. The expression of the protein C deficiency was mainly recurrent superficial and deep iliofemoral vein thrombosis and pulmonary embolism. The protein C deficiency was also expressed as generalised peritonitis due to massive messenteric vein thrombosis, cavernus sinus, renal vein thrombosis and priapism. In one of these families, five members died of intra-abdominal thrombosis before the age of 40. A compensated diffuse intra- vascular coagulation syndrome was observed during massive thromboembolic attacks as evidenced by high levels of D-Dimer (≥5000ng/ml). The treatment of choice was heparin or urokinase (with the exception of one patient), followed by heparin and fresh frozen plasma. Long term prophylaxis was LMW heparin or low dose warfarin plus stromba. The one patient who did not respond to the thrombolytic treatment with urokinase was found to have in his plasma a high titre of inhibitor against urokinase and prourokinase. This patient responded to streptokinase treatment. D-Dimer levels in these patients in non-crisis state were raised and proportional to the degree of the protein C deficiency.
NEONATAL HOMOZYGOUS PROTEIN C DEFICIENCY. PROBLEMS IN DIAGNOSIS AND MANAGEMENT
Authors: Civantos F , Kent J , Pegelow C H
Published: June 2019

A newborn with a large rapidly necrotizing hematoma in the right buttock had initial coagulation studies suggestive of disseminated intravascular clotting. Negative cultures, development of other ecchymotic lesions in the scalp, eyelid, and elbows and response to fresh frozen plasma allowed the clinical diagnosis of homozygous protein C deficiency that was confirmed by protein C levels of .00 U/ml immunological and .085 U/ml by coagulation assay. Immunologic.protein C assays in the family showed: .41 U/ml in the mother, .38 U/ml in the father, and .55 U/ml in the paternal grandfather with similar functional assay values. CT scans showed thrombosis of dural venous sinuses with bilateral infarcts and possible subarachnoid hemorrhage resulting in rapidly developing hydrocephalus. Cataracts and synechiae developed in both eyes as a result of hemorrhage at birth. Further episodes of thrombosis and hemorrhage were prevented by administration of fresh frozen plasma every 12 hours. Problems ensued with development of hyper-proteinemia, hypercalcemia and hyperphosphatemia. A shunt to control the hydrocephalus became infected as did the catheter for fresh frozen plasma administration. Coumadin administration concurrent with fresh frozen plasma administration was difficult to regulate; phenobarbital given for subclinical status epilepticus interfered with Coumadin. Factor VII assays were used to regulate the concomitant administration of Coumadin and fresh frozen plasma. At 8 months a new episode of purpura fulminans caused the patient's demise. Skin biopsy of the lesions at birth and autopsy sections of new skin lesions showed thrombosis of subcutaneous adipose tissue veins with surrounding hemorrhage. The pathologic and dermatologic findings were identical to those of Coumadin-induced skin necrosis.
DEFECTS OF VITAMIN K-DEPENDENT FACTORS IN CA(11)-STABILI ZED STRUCTURE
Authors: Poort S R , Krommenhoek-van Es C , van der Linden I K , van Tilburg N H , Bertina R M
Published: June 2019

Vitamin K-dependent (anti)coagulation factors (factor II, VII, IX, X protein C and S) undergo a conformational transition upon binding of Ca(II), which is a prerequisite for their normal function. Abnormalities in these properties occur during vitamin K deficiency or treatment with anti vitamin K drugs and in some genetic variants of coagulation factors. Immunological assays utilizing antibodies against the Ca(II)-stabilized structure are useful to detect such abnormalities.

Starting from specific rabbit antisera antibody populations specific for the Ca(II)-dependent conformation of factor II, VII, IX, X and protein C and S were isolated using immuno-affinity procedures. Subsequently immunoradiometric assays specific for the Ca(II)-dependent (Ca(II)Ag) and Ca(II)-independent (NonCa(II)Ag) conformations of the different proteins were developed. These assays were used for the analysis of plasmas of patients stably treated with oral anticoagulants; Ca(II)Ag, NonCa(II)Ag and their ratio were measured as function of the intensity of the treatment (INR 2.4 to 4.8). The same parameters were measured in plasmas of patients with hereditary coagulation disorders. After treatment with oral anticoagulation with an antivitamin K drug reduced ratios of Ca(II)Ag/-NonCa(II)Ag were observed for factor II, VII, IX, protein C and protein S. However, the actual degree of reduction and its dependence on the intensity of treatment varied for the different vitamin K-dependent proteins. In general Ca(II)Ag levels correspond nicely with the procoagulant activity of the concerning proteins. These data provide indirect evidence for the existence of abnormal (non and/or subcarboxylated) forms of the vitamin K-dependent proteins during oral anticoagulant treatment.

Genetic variants with a mutation in one of the sites involved in the formation of the Ca(II)-s tab i1ized structure could be detected for factor IX, factor VII and factor II. However, the extent of reduction of the ratio Ca(II)Ag/-NonCa(II)Ag differed considerably in those variants.
Purification and Characterization of Bilokinase, a Biliary Plasminogen Activator
Authors: Oshiba S. , Ariga T.
Published: June 2019

A biliary plasminogen activator was first found in human and animal bile by Oshiba et al. and termed bilokinase (Jap. J. Physiol. 19, 212, 1969). Purification of this activator from bovine bile was later achieved through a six-step procedure consisting of a new extraction method using (NH4)2SO4/acetone under controlled condition, followed by chromatography with SP-, G-100 and CM-Sephadex. The highly purified preparation exhibited a single band in disc electrophoresis. A sedimentation velocity study gave S20,w value of 3.32 S. The molecular weight was estimated at 58,000 from the elution volume/void volume ratio. A comparative study with urokinase showed a different mode of action for its substrate specificity toward AGLMe (Km) and its behavior toward (AMCHA (Ki). Amino acid analysis of our preparation indicated a higher lysine but lower tyrosine content than in human urokinase (W. F. White et al., Biochem. 5, 2160, 1966), among some other differences. A distinct immunological dissimilarity between bilokinase and urokinase was eluciadated by Ouchterlony’s method and from inhibition with anti-bilokinase serum.
Pathobiology of Human Fibrinogen. Binding site for Staphylococci on Aα and Bβ chains of Fibrinogen Molecule
Authors: Hawiger J. , Hammond D. K. , Timmons S.
Published: June 2019

The ability of human fibrinogen to interact with some staphylococci to cause a non-enzymatic clumping reaction has been used to detect fibrinogen and its derivates in blood and body fluids. We studied localization of the binding site for staphylococci on human fibrinogen using 125I-fibrinogen after reduction and alkylation of polypeptide chains, CNBr-treatment, and limited plasmin digestion. This combined approach revealed that the staphylococci-binding site is located on Aα and B β chains involving C-terminal portion but not N-terminal disulfide knot, γ chain has no binding site for staphylococci. Abnormal fibrinogens with defects in the N-terminal portion interacted normally with staphylococci. The degree of binding paralleled the extent of clumping reaction between staphylococci and 125I-fibrinogen or its plasminderived fragments. However, monomeric Scarboxymethyl Aα and B β chains showed a high degree of binding without concomitant clumping suggesting requirement for dimeric form of polypeptide chains for clumping. Conclusion: Aα and Bβ chains but not γ chain have homologous regions in terms of staphylococci-binding site on C-terminal portion responsible for clumping reaction. Thus, staphylococci can be used to measure abnormal fibrinogens with defects in the N-terminal region.

(Supported by NIH Grants and VA.)
Studies of the Interaction of Platelets with Highly Purified Macromolecules of the Arterial Wall
Authors: Ordinas A. , Hornebeck W. , Robert L. , Caen J.
Published: June 2019

Collagen, structural glycoproteins and elastin are the major components of the arterial wall. The first fibrillar component is well known as an aggregatin agent for platelets. Little information is available on the possible role of elastin or structural glycoproteins in this respect. The purification and characterization of these two macromolecules are described as well as a new methodology for the study of their interaction with platelets using affinity columns, with Sepharose 2B as an inert support. The elution pattern of platelets on such columns suggests an interaction of defined platelets with these macromolecules. The results obtained by this method complete and extend those obtained with the native aorta preparation (Baumgartner's technique). The results obtained enabled us to propose a molecular mechanism for the interaction of platelets with the subendothelium.
Effect of Acetyl Salicylic Acid on Platelet Function in Yivo
Authors: Hassanein A. A. , Afifi N. , Badr M.
Published: June 2019

The ingestion of a single dose of 300 mg. acetyl salicylic acid (ASA) cause after two hours, significant diminution in ADP-induced platelet aggregation and increase in platelet disaggregation. The calcium-induced platelet aggregation test, a turbidimetric modification of the Chandler tube technique, showed significant prolongation in the onset of aggregation and in the clotting time. A test based on the contact product forming activity of platelets showed inhibition after ASA intake.

It is suggested that acetyl salicylic acid in addition to its inhibitory effect on platelet release reaction, also affects primary aggregation by ADP and possibly interferes with the ability of platelets to activate the contact system of the intrinsic blood coagulation pathway.
Daunomycin and Platelet Function
Authors: Whaun J. M. , Dundas J. E. , Clarke H.
Published: June 2019

Daunomycin, an antimetabolite antibiotic used in the treatment of acute leukemia, is highly cytotoxic to both normal and malignant cells. Thrombocytopenia occasionally noted with its use, has usually been attributed to its toxic effect on the bone marrow. Citrated platelet-rich plasma (PRP) from normal volunteers was tested in vitro in the aggregometer after addition of either aggregating agent, collagen, (0.083 mg/ml PRP), or varying concentrations of daunomycin, or both. Other aliquots were exposed to daunomycin or saline for 90 seconds before fixing in glutaraldehyde. The remainder, labelled with 0.5 μM 2−14C-5-hydroxytryptamine binoxalate (14C-5-HT), 27.5 c/mole for 15 minutes, was tested for 14C-5-HT release before and after exposure (90 sees. 37° C) to collagen of final concentration 0.04 mg/ml.

At a concentration 0.001 mg/ml PRP, daunomycin was associated with decreased response of platelets to collagen with decreased 14C-5-HT release. At 0.02–0.04 mg/ml daunomycin was associated with vacuole formation, loss of dense bodies as well as platelet lysis as well as liberation of newly absorbed 14C-5-HT. Electron micrographs of platelets exposed to higher doses of daunomycin showed platelet swelling, vacuole formation, swollen mitochondria and granules and interruption of the trilaminar membrane.
Platelet Dysfunction Induced by Parenteral Administration of Carbenicillin and Ticarcillin
Authors: Johnson G.J. , White J. G.
Published: June 2019

Carbenicillin treatment of humans has resulted in impaired platelet function occasionally accompanied by hemorrhage. Because of their therapeutic potential as anti-thrombotic agents, we have studied carbenicillin (CARB) and ticarcillin (a-carboxy-3-thienylmethyl penicillin) (TIC) effects on platelet function in the dog. Studies of platelet aggregation (AGG), adhesiveness (ADS), factor 3 availability (PF3), bleeding time (BT), electron microscopy (EM), and serotonin (5HT) and adenine nucleotide (AN) content were performed sequentially in dogs given CARB or TIC. Progessive impairment of platelet function was observed with increasing doses from 250 to > 1250 mg/kg/day. Doses of 250 to 750 mg/kg/day resulted in mild to marked impairment of ADP induced AGG, markedly decreased ADS, and moderately prolonged PF 3 clotting times. Collagen induced AGG was impaired and the BT was prolonged only at doses > 750 mg/kg/day. Platelet dysfunction was evident at 4 hr., significant at 24 hr., and maximal at 72 to 96 hr. with little further progression up to 14 days despite continued treatment. At equivalent doses CARB was slightly more inhibitory than TIC. EM, 5HT, and AN did not change significantly. Platelet function was significantly improved 24 hr. after treatment was discontinued, and progressive improvement continued for 5 to 14 days.

These studies demonstrate that CARB and TIC induce a dose-related impairment of platelet function. They suggest that these drugs interfere with the platelet release reaction. They indicate that CARB and TIC are promising anti-thrombotic agents which merit further study.
Prevention of Ischemic Cerebro Vascular Accident; A Long Term Clinical Trial with Vasodilator an dAntiaggregatin Drugs
Authors: Guiraud B. , David J. , Géraud G. , Boneu B. , Biermé R. , Rascol A.
Published: June 2019

The purpose of this study is to determine whether antiaggregating drugs are more effective than vasodilators in the prevention of ischemic cerebrovascular accidents.

To date, 350 patients are randomly divided into 3 groups and treated as follows: group 1 – hydergin alone (4.5 mg/day); group 2 – hydergin (4.5 mg/day) plus aspirin (1 g/day); group 3 – hydergin (4.5 mg/day) plus aspirin (1 g/day) plus dipyridamole (15 Omg/day).

Only those patients with a history of transient ischemic attacks or a regressive stroke with minimal sequellae are included. Patients with coagulopathy, long term anticoagulant or anti-inflammatory treatment, valvulopathy or peptic ulcus are excluded. Cerebral angiography is performed only when indicated. All patients are clinically examined three times a year with platelet aggregation performed at each visit to control correct drug intake.

This study, begun on the june 1st 1973, will be followed for a three years period once a total sample population of 400 patients is reached.

A preliminary interpretation of the data will be completed.
A New Assay for the Measurement of Total Progressive Antithrombin
Authors: Lane Janet L. , Bird Prudence , Rizza C. R.
Published: June 2019

A new rapid method for assaying total antithrombin activity has been developed based on the inactivation of thrombin incorporated into an agarose gel, during the radial diffusion of plasma in the gel. The area of thrombin inactivation is subsequently observed by the coagulation of fibrinogen in a separate agarose gel layer poured over the thrombin gel. The method is described in detail and its accuracy assessed with respect to other antithrombin assays. Using specific antisera to α2-globulin (antithrombin III), α2-macro-globulin and α1-antitrypsin, total antithrombin activity measured by this assay consisted of 47% α2-globulin, 29% α2-macroglobulin and 26% α1-antitrypsin.
Defibrinogenation Procedures for Antithrombin III (AT-III) Biological Assay
Authors: Tonolli M. C. , Donati M. B.
Published: June 2019

Since fibrinogen has itself an antithrombin activity, defibrinogenation of the sample is a prerequisite for AT-III determination. Five different defibrinogenation procedures were tested: heat precipitation for either 3 or 10 minutes, ristocetin precipitation, clotting with either Reptilase-R or Botropase-R. AT-III was measured as biological activity according to Howie et al. (Br. J. Haemat. 25, 101, 1973) and assayed immunologically by radial immunodiffusion. Defibrinogenation was complete with all the procedures used (< 16 μg/ml F. R.-antigen being left). In the samples defibrinogenated by ristocetin, the biological assay could not be performed, since some ristocetin remained in the supernatant and provoked a rapid precipitation of the substrate fibrinogen used for the assay. The possibility that Botropase-R or Reptilase-R had themselves some antithrombin activity was preliminarly ruled out. In samples definbriogenated by heating (3 min), Reptilase-R or Botropase-R the same biological AT-III activity was found; in addition, no differences in immunological reactivity of the protein were observed as compared with the corresponding-plasma before defibrinogenation. In contrast, a statistically significant (P < 0.001) reduction of both biological and immunological AT-III activity was found in the samples defibrinogenated by heating (10 min), as compared with the other 3 procedures. These results indicate that Reptilase-R and Boropase-R are suitable reagents for defibrinogenation of plasma prior to AT-III assay; heat precipitation can also be used, provided the duration of the incubation period is accurately controlled.

(Supported by Grant N. 73.00400.04 of the Italian Research Council (C. N. R.).)
The Effect of Divalent Cations on the Interaction between Thrombin and Antithrombin
Authors: Osamo N. O.
Published: June 2019

The importance of calcium irons for the action of pro-coagulant blood clotting factors has been known since the time of Morawitz’s classical theory and the practical application of this knowledge is demonstrated in the use of anticoagulants for the collection of blood used in laboratory investigations. The necessity for cations in the interaction between the pro-coagulant factors and their inhibitors has not been described. We now find that during the isolation and purification of antithrombin from normal plasma, the use of dialysis tended to diminish or even abolish the activity of this inhibitor. However, when the dialysate was reintroduced into the dialysed plasma or fraction containing the antithrombin, the activity was restored, although the dialysate itself showed no antithrombin activity. On reintroduction of divalent cations in place of the dialysate, the activity was also restored. It seems therefore, that these divalent cations are necessary for the interaction of thrombin and antithrombin. Although the exact mechanism of action of these cations has not been completely elucidated, it would appear that they act possibly by the removal of charges or by facilitation of bonding between the two reactants.
Modification of the Hyperacute Reaction in the Rat by Sulphinpyrazone
Authors: Jamieson S. W.
Published: June 2019

It has been found that guinea-pig hearts are rejected in hyperacute fashion by the rat. By using inbred strains this model has been found to be markedly reproducible. Electron microscopic studies show that the platelet plays a critical role in the rejection process. An important point is that endothelial cell damage morphologically occurs subsequent to platelet contraction.

Flow studies demonstrate that flow through the coronary arteries ceases abruptly between 2 and 3 minutes; this co-inciding with platelet aggregation in the vessels. The heart continues to beat for 10–12 minutes, and then suffers an ischaemic death.

30 transplants were performed as controls to fully monitor flow, E. C. G. and microscopic events during the reaction.

Ten transplants were performed in rats pre-treated with 150 mgs/kg. Sulphinpyrazone orally daily for 6 days and then transplanted, and ten were performed in rats transplanted and then given 400 mgs/kg sulphinpyrazone I–V before release of the clamps preventing revasularisation of the heart.

It was found that rejection time was not significantly changed from the controls (12–14 minutes), but flow studies showed that the coronary circulation remained patent for 5–6 minutes, i.e. twice the time of the control studies, with subsequent delay of E. C. G. changes.
A Radioimmunoassay for Human Platelet β-Thromboglobulin
Authors: Ludlam C. A. , Bolton A. E.
Published: June 2019

β-thromboglobulin (βtg) is a protein recently isolated from human platelets. A radioimmunoassay for βtg has been established.

Using this assay, conditions have been defined for the preparation of platelet poor plasma, so as to minimise the liberation of βtg during manipulations in vitro. Platelet poor plasma prepared from whole blood, collected in EDTA, prostaglandin E9, and theophylline, and centrifuged at 0°C., contained 0.019±0.0075 μg ml−1 (mean±l SD). Serum prepared from clotted whole blood (in which platelets had undergone the release reaction) had a concentration of 17.4±6.3 μg ml−1, while plasma from platelet transfusion concentrates contained 247.0±120.3 μg ml−1. There was no increase in βtg concentration when platelet poor plasma was clotted by the addition of calcium. The presence of a 1000-fold difference between plasma and serum concentrations, the observed release following collagen induced platelet aggregation in vitro, and the observations that other human tissues contained only trace amounts suggests that βtg is unique to platelets.

Preliminary clinical studies have shown that patients with acute arterial thrombosis and others with prosthetic heart valves have raised plasma concentrations. It is possible, therefore, that this assay has potential uses for studying the platelet release reaction in vitro, and also the identification of individuals with excessive platelet sequestration in vivo.