bluuebunny/crossref_metadata_2025 huggingface dataset:
Familial Primary Fibrinolysis
Authors: Currimbhoy Z.
Published: May 2019

This study reports on a unique family with primary fibrinolysis. A 31-year-old female had hematuria of one month’s duration. She had a previous episode of hematuria lasting 3 weeks and a hysterectomy for menorrhagia. Her 10-year-old daughter bled from the gums at the age of 9 months for one week. All clotting factors (including Factors VIII, V and XIII) were normal, except Fibrinogen. The mother’s Fibrinogen varied from 110 to 165 mg%, and the daughter’s from 90 to 146 mg%. The diluted Thrombin Time and Reptilase times were normal. The most striking and constant defect was in the fibrinolytic system. These studies were performed over a 4 month period. Euglobulin clot lysis, done on the mother repeatedly and on the daughter twice, were abnormally short, varying from 60 to 100 minutes, with FSP>10 and <40 ug/ml or <10 ug/ml. Active lysis was present on fibrin plates. α2 macroglobulin, α1 antitrypsin and Antithrombin III were normal, as was plasminogen immunologically. Platelet count and functions were normal. The maternal grandmother bled profusely twice after two gynecological procedures and once needed a blood transfusion. She was then put on birth control pills. Work-up on the pill revealed a Fibrinogen of 217 mg%, but no clot formed when attempting to evaluate euglobulin lysis. Off the pill, Fibrinogen was 167 mg%, euglobulin clot lysis 30 mins,, and there was a zone of lysis on the fibrin plate.

The congenital loss of an inhibitor to plasminogen activator could explain these results.
Release of Fibrinolytic Enzymes by Macrophages in Response to Soluble Fibrin
Authors: Sherman L. A. , Lee J. , Stewart C. C.
Published: May 2019

In previous data (J. Exp. Med. 147:76,1977), we have demonstrated that soluble fibrin/fibrinogen complexes are bound to the plasma membrane of guinea pig peritoneal macrophages. This binding is largely irreversible and is not a result of phagocytosis. We have extended our studies to examine the response in vitro of peritoneal macrophages to soluble fibrin/fibrinogen complexes. Unstimulated mouse macrophages were collected by peritoneal lavage and 5–60 μg of soluble fibrin/fibrinogen complexes placed into tissue culture dishes containing the unstimulated cells. Aliquots of the media were collected at 24, 48 and 72 hours. The cell-free media contained increasing amounts both of plasminogen activator and an enzymatic activity which resulted in fibrin and fibrinogen proteolysis independent of the amount of plasmingoen present. The major proteolytic activity was due to the non-plasminogen dependent enzyme. Similar enzymes were released from peritoneal macrophages stimulated in vivo. The plasminogen activator enzyme had a low molecular weight comparable to that previously reported by Unkeless et al, with in vivo stimulation. Other coagulation moieties, such as plasmin and α-2 macroglobulin plasmin complexes did not result in release of the macrophage proteolytic enzymes. The results suggest that the previously described release of fibrinolytic enzymes after thioglycolate injections, may also result from the more pathophysiological stimulation by soluble fibrin/fibrinogen complexes. Release of these enzymes from phagocytic cells may be important, not only in blood clearance of soluble fibrin/fibrinogen complexes, but as part of thrombus reabsorption and wound healing.
Thrombolytic Effect of a Proteolytic Enzyme from Aspergillus Ochraceus, Awegillase(R)
Authors: Landmann H. , Markwardt F.
Published: May 2019

The mould Aspergillus ochraceus produces and liberates into the culture medium a proteolytic enzyme. It was highly purified and obtained in crystalline form by ethanol-calcium precipitation. The enzyme (molecular weight approximately 20,000) hydrolyses typical trypsin and chymotrypsin substrates as well as proteins, such as casein, haemoglobin and fibrin. It is inhibited by blood plasma antiproteinases. In animal experiments, the enzyme shows relatively low toxicity as well as antithrombotic and thrombolytic effects, even at doses lower than the plasma antiproteinase level. Experimentally produced clotting and deposition thrombi in peripheral veins and arteries as well as in coronary arteries are removed by proteolytic degradation. Based on first clinical studies, the enzyme, Awegillase(R), is considered a suitable tool for both local and systemic thrombolytic therapy.
Physicochemical and Biochemical Properties of Commercially Available Urokinase Preparations
Authors: Nishida M. , Nishimaki H. , Miyake S. , Suyama T. , Morisue S.
Published: May 2019

Comparative studies were made on eleven urokinase preparations commercially available. Analysis by both gel filtration and sodium dodecyl gel electrophoresis revealed the variety of molecular forms of the activator in the preparations showing molecular weight approximately 54,000 (A), 47,000 (B) and 34,000 (C). Starch gel electrophoresis at pH 8.8 indicated that (B) and (C) moved to anode whereas (A) and the active component of fresh urine slightly moved to the cathod. Proteolytic digestion of (A) produced the same component as shown by (B) and (C) on starch gel electrophoresis. Plasminogen activating activity of (B) and (C) was found to be less than that of (A) when measured by the procedure of CTA fibrinolytic method with the physiological blood level of plasminogen. The present data suggest that variety of molecular form in the preparation may be due to the difference of purification procedure in view of proteolytic degradation of the enzyme, and (A) seems to be the naturally occuring type of urokinase in urine.
Prothrombin Biosynthesis: The Vitamin K-Dependent Carboxylase
Authors: Suttie J. W. , Lehrman S. R. , Rich D. H. , Whitlon D. S.
Published: May 2019

We have shown that the pentapeptide Phe·Leu·Glu·Glu·Val serves as a substrate for a vitamin K-dependent liver microsomal carboxylase that converts peptide-bound glutamyl residues to γ-carboxyglutamyl residues. Requirements of the solubilized microsomal system for peptide car-boxylation have been determined and are similar to requirements for prothrombin precursor carboxylation. Additional peptides have now been synthesized, and the available data suggest that specificity of the vitamin K-dependent carboxylase involves both macromolecular and active-site recognition sequences. Inhibition of the in vitro carboxylation system by the anticoagulant Warfarin has now been studied in detail. Dithiothreitol stimulates carboxylase activity and replaces the requirement for NADH as a reductant needed to form the hydroquinone. Warfarin is a potent inhibitor of the DTT driven carboxylase reaction in intact microsomes, but a much poorer inhibitor when NADH is used as a reductant. In a microsomal system with DTT (but not NADH) as a reductant, vitamin K epoxide is an active form of the vitamin. In a Triton X-100 solubilized system; NADH is required, Warfarin is ineffective as an inhibitor, the epoxide is not an active form of the vitamin, and the conversion of vitamin K epoxide to the vitamin cannot be demonstrated. These data support the hypothesis that Warfarin inhibits vitamin K activity through an effect on epoxide reduction and suggests that this reaction is driven by a nonpyridine nucleotide pathway which, in vitro, can use DTT instead of an unidentified physiologically active reducing agent.
Multiple Molecular Forms of Factor VIII Antigen in Normal Individuals and Von Willebrand’s Disease Patients
Authors: Zimmerman T. S. , Kimball J. R. , Edgington T. S. , Abildgaard C. F.
Published: May 2019

Factor VIII antigen is present in normal individuals in multiple molecular forms which can be separated according to size. The intermediate and larger forms preferentially bind to platelets in the presence of ristocetin. In order to evaluate the possibility that Factor VIII antigen forms of large size may be an artifact of in vitro aggregation, we have ultracentrifuged plasma on a 20% sucrose cushion at 37°C for 10 min at 254,000 xg (peak). The rate of clearing of Factor VIII antigen was compared to that of IgM, fibrinogen, IgG, α1-antitrypsin and the S rate calculated to be between 15 and 18. These results indicate that Factor VIII-related antigen forms of high S exist even when plasma is maintained at physiological temperature and analyzed with minimal delay, suggesting that these large molecular forms also exist as such in vivo.

Two types of von Willebrand’s disease (vWd) have been identified according to size of Factor VIII antigen forms present in plasma. In Type I all forms of Factor VIII antigen are present but are decreased in quantity. In Type II large forms are missing and smaller forms are present in normal or increased quantities. Factor VIII antigen was isolated from plasma of one patient with Type I and two patients with Type II vWd by counter Immunoelectrophoresis. The Factor VIII antigen was then reduced and electrophoresed on SDS-containing polyacrylamide gels. The presence of carbohydrate was evaluated by staining with perchloric acid-Schiff’s reagent (PAS). The 210,000 MW Factor VIII antigen subunit from each patient was PAS-positive. Though subtle changes in carbohydrate content or composition could not be evaluated by this technique, a total defect of glycosylation is unlikely in this small sample of vWd patients.
The von Willebrand Pig as a Model for Atherosclerosis Research
Authors: Fuster V. , Bowie E. J. W. , Lewis J. C.
Published: May 2019

The aortas of 11 pigs with homozygous von Willebrand’s disease (vWd) were compared with those of 11 normal pigs, all aged 1 to 3 years. Six of the controls exhibited multiple arteriosclerotic plaques over 2 mm. in diameter with intimal thickenings of 63 to 130 microns. In contrast, none of the vWd pigs had multiple plaques; one had a single lesion over 2 mm. in diameter.

Ten additional pigs, 5 controls and 5 with homozygous vWd, were placed on a 2% cholesterol diet for 6 months, beginning at the age of 3 months. Four of the controls developed aortic arteriosclerotic lesions exceeding 7.5% of the entire surface. Intimal thickness ranged up to 370 microns. In contrast, 4 of the vWd pigs developed lesions not exceeding 0.5% of the aortic surface; the fifth vWd pig had arteriosclerotic lesions involving 7.3% of the aortic surface.

The aortas of the vWd pigs did stain with Evans blue dye injected antemortem, and they exhibited fatty infiltration in the intima. By electron microscopy, severe endothelial damage was apparent, but there was no intimal proliferation.

The vWd pig seems to be an ideal model for arteriosclerosis research and the possible relationship of our findings may be related to impaired platelet-arterial wall interaction in vWd.
Antibodies as a Cause for Platelet Functional Defects
Authors: Caen J. P. , Levy-Toledano S. , Tobelem C. , Michel H. , Nurden A. T. , Legrand C. , Degos L.
Published: May 2019

The Luc. antibody occurring in a polytransfused thrombasthenic patient is an IgG which agglutinates all human platelets except thrombasthenic ones. At sub-agglutinating doses, ADP, Adrenaline, Collagen, Thrombin and arachidonic acid-induced aggregations but not ristocetin and bovine factor VIII mediated platelet aggregations are inhibited. It does not modify ADP binding to normal human platelet membranes. The antibody strongly reduced thrombus formation on rabbit aorta subendothelium. Its activity is consumed after incubation with normal human platelet membranes but not with thrombasthenic ones. It inhibits thromboxane formation with all inducers but not with arachidonic acid and PGG2. This antibody reacts with a component present on human platelet membranes 120.000 MW.

The Por. antibody occurring in a polytransfused Bernard-Soulier patient is an IgG which agglutinates all human platelets except those of Bernard-Soulier and thrombasthenia. The activity is consumed after incubation with normal and thrombasthenic platelet membranes but not with platelet membranes of Bernard-Soulier patients. At sub-agglutinating doses only ristocetin, bovine factor VIII aggregation and adhesion of human platelets to rabbit aorta subendothelium are inhibited. Purified neuraminidase, chymotrypsin or trypsin destroy the reaction of normal platelets with this antibody. This IgG reacts with a component of human platelet membrane 155.000 MW.
Is Fibrinolysis a Continuous Low Level Mechanism in Normal Adults?
Authors: Plow E. F. , Edgington T. S.
Published: May 2019

Continuous fibrino(geno)lysis by plasmin has been suggested as a primary pathway for fibrinogen catabolism and as a means of compensating for ongoing low levels of blood coagulation and fibrin formation. The cleavage-associated neoantigen, fg-Dneo, provides an incisive immunochemical marker for assessment of this hypothesis since it is expressed in an equimolar fashion by all major physiologic cleavage fragments (X, Y and the D:E complex) of fibrinogen and fibrin. In previous studies (J. Clin. Invest. 52:273, 1973), fg-Dneo expression was not detected in normal plasma, but the sensitivity of the described assay was not sufficient to exclude low levels of these derivatives. In the present study, utilizing sequential radioimmunoassay procedures, preincubation of the competing antigen with anti-fg-Dneo prior to addition of the 125I-ligand (fg-D), the sensitivity of the assay exceeded 1x10-11M (<2 ng fg-X/ml). In this system, 20 plasmas from healthy adults were found to contain no detectable fg-Dneo activity. On the basis of assay sensitivity, less than one molecule of degradation product is present per 100,000 fibrinogen molecules in normal plasma. In separate studies, it was established that the initial cleavage of the amino-terminal aspects of the Bβ chain of fibrinogen were required for exposure of fg-Dneo. Aα. chain cleavage alone was insufficient as high solubility fibrinogen fractions 1–8 and 1–9 failed to express fg-Dneo. On this basis, it is concluded that fibrinolysis and fibrinogenolysis sufficient to produce fragment X or later plasmic derivatives do not represent significant normal physiological pathways.
The Biphasic Effect of Sulfinpyrazone on Platelet Function
Authors: Buchanan M. R. , Rosenfeld J. , Hirsh J.
Published: May 2019

Experiments performed in vitro have shown that the effect of sulfinpyrazone (SUL) on platelet function is associated with inhibition of platelet prostaglandin synthesis. We have studied platelet aggregation in vivo, plasma-drug levels and platelet MDA production in rabbits after treatment with SUL. At a dose of 100mg/kg, collagen-induced platelet aggregation in vivo was inhibited by 44% at ½ hr, 20% at 4 hr and then increased to a maximum inhibition of 57% at 18hr. This latter effect was achieved when there was no detectable SUL in the plasma (<0.5ug/ml). Platelets prepared from rabbits given 100mg/kg SUL either 2 or 8 hr beforehand were washed, labelled with 51Cr and injected into normal rabbits and platelet aggregation in vivo was measured. The response to collagen of platelets exposed for 2 hr to SUL was normal, whereas the response of platelets exposed for 18 hr to SUL was inhibited by 32%. MDA production by platelets incubated for 60 min in PPP prepared from rabbits which had been treated with either 10mg/ kg 1 hr beforehand or 100mg/kg 18 hr beforehand, was measured.(The SUL levels of the former and the latter plasmas were 4.4 and <0.5ug/ml respectively.) Platelet MDA production was inhibited 15% after incubation in the 10mg/kg-l hr PPP and 32% after incubation in the 100mg/kg-18 hr PPP (p<0.0001).

It is concluded that SUL has a biphasic effect on collagen-induced platelet aggregation in vivo. Both effects are associated with inhibition of MDA production but the initial effect is reversible with washing and less marked than the secondary effect which is irreversible and could be due to a metabolite still present in the plasma at 18 hr.
Inhibition of Platelet Thrombosis by N-Acetyl Neuraminic Acid (NANA)
Authors: Kovács Iren B. , Born G. V. R.
Published: May 2019

In normal venules of hamster cheek pouch, platelet thrombogenesis is inhibited by intravenous neuraminidase or by NANA at concentrations (up to 50 μg/ml) produced by neuraminidase from endogenous sources (A. Atherton & G. V.R. Born,1973; J. Physiol., 234, 66P). To find out whether this inhibition also occurs in damaged vessels, venules in hamster cheek pouch or rat mesoappendix were irradiated from a HeNe laser for 5 sec. every min., after an intravenous injection of Evan’s blue (T1824) to absorb the laser energy (I. B. Kovács, A. Tigyi-sebes, K. Trombitas & P. Görög, 1975; Microvasc. Res. , 10, 107). In hamsters, intravenous NANA (10 μg/g body weight) decreased thrombus growth rate after 3 to 9 min. by about 72%. In rats the same dose inhibited thrombogenesis completely for up to 30 min.; 5 μg/g decreased the growth rate. NANA inhibited whether injected at pH 2.3 or 6.0.Under these conditions NANA did not alter blood pH, platelet concentration, or blood flow. Thrombus growth was not inhibited by D-glucuronic acid or by the β-methyl glycoside of NANA at the same molar concentrations as NANA. Therefore, inhibition by NANA of platelet thrombogenesis occurs in damaged as in undamaged vessels.
The Characterization of Pig Platelet Membrane Proteins: Studies on Membrane-Associated Actin and the Surface-Associated Phosphodiesterase Activity
Authors: Taylor D. G.
Published: May 2019

Analysis of the polypeptides of a pig platelet surface membrane fraction by SDS-polyacryl-amide gel electrophoresis revealed the presence of approx. 12 components (molecular weight range 12000–200000).A component of 43–45,000 daltons was particularly prominent and this has been identified as actin, and shown to be similar in many respects to the cytoplasmic protein. The membrane-associated actin has been isolated as a pure band by preparative SDS-polyacrylamide gel electrophoresis and analysis of its amino acid composition, although very similar to that of actin purified from whole platelets, showed a much lower content of 3-methylhistidine (approx. 0.05 residues/mole compared with 0.62 residues/mole). The surface membrane-associated phosphodiesterase (towards bis-(p-nitropheny1) phosphate) has also been partially characterised. This enzyme activity can be completely solubilised from the membrane using 0.5% Triton X-100, and can then be isolated as a single peak on Sepharose 4B. The separated enzyme fraction showed considerable purification over the original membrane as illustrated by SDS-polyacrylamide gel electrophoresis with the prominent component being the 95,000 dalton polypeptide. Our studies have also shown that this phosphodiesterase is inhibited competitively by ADP and ATP.
vWAgII: A New von Willebrand’s Disease Antigen
Authors: Montgomery R. R. , Zimmerman T. S.
Published: May 2019

Factor VIII-related antigen (vWAg I) is a protein of high molecular weight which co-purifies with Factor VIII procoagulant activity and von Wiflebrand Factor under a variety of conditions. It is decreased or absent in most cases of von Willebrand’s disease (vWD). We now report the existence of a second antigen (vWAgII) which is absent from the plasma of individuals (3/3) with severe vWD but present in normal amounts in the plasmas of individuals (8/8) with moderate or variant vWD. vWAgII is present in normal platelets and is released from them during clotting. However, it is also present in platelet-free plasma from normal individuals which has been collected in a variety of protease inhibitors including benzamidine, heparin, Trasylol, PMSF and soybean trypsin inhibitor suggesting that it circulates in plasma in vivo. The plasma from a patient with intravascular coagulation was found to have increased vWAgII.

vWAgII is detected by antisera raised against void volume fractions of American Red Cross Factor VIII concentrates subjected to agarose gel chromatography. Antisera raised against highly purified vWAgI do not detect vWAgII. Crossed immunoelectrophoretic analysis shows vWAgII to have a more anodic mobility than vWAg I. II exhibits no immunologic cross-reactivity with intact vWAgI nor with vWAg I fragments produced by plasmin, a-chymotrypsin, trypsin or elastase. Though the absence of both antigens from the plasma of individuals with severe vWD suggests a biologic linkage, immunologic evidence for the origin of these two species from a common precursor has not been demonstrated.
Urea Denaturation of Antihemophilic (AHF) and von Willebrand (vWF) Factors in the Presence and Absence of Selected Amino Acids
Authors: Harris R. B. , Newman J. , Johnson A. J.
Published: May 2019

AHF and vWF are functionally independent activities in human plasma and a large body of data suggests that they may be different proteins, including a striking difference in the rate and extent of denaturation by urea. Thus, concentrations of urea below 1.5M resulted in 100% loss of vWF, 70% loss of AHF and 35% loss of Factor VIII related antigen (VIII Ag) in two hours, while 3M urea caused a loss of 100% vWF, 90% AHF and 45% VIII Ag in 30 hours. Full activity of urea denatured vWF and VIII Ag could be restored by dialysis of the urea while the AHF activity returned to about 75% of the initial value. Substitution of acetamide for urea resulted in only partial denaturation of the AHF (50% of that obtained with urea) suggesting that urea may interact with these proteins through the formation of bifunctional hydrogen bonds. Lysine and structural analogs of lysine with free amino groups prevented urea denaturation of the AHF but urea denaturation of vWF was not prevented by these amino acids. These data suggest that loci i essential for the biological activity of AHF, vWF and VIII Ag are denatured by direct interaction with urea. Since VIII Ag and AHF are more resistant and slower than vWF in their reaction with urea, other bonds or groups are also important. The differences in denaturation, renaturation and protection against urea denaturation of AHF and vWF by selected amino acids could be explained if the reactive sites for each activity were located on different molecules as suggested by other in vitro and in vivo observations or were remote from one another on one molecule.
Simultaneous Measurement of Platelet Factor 4 (PF4) and β-Thromboglobulin (βTG) Release and Fibrinopeptide A (FPA) Cleavage
Authors: Kaplan K. L. , Nossel H. L.
Published: May 2019

Platelet activation and fibrin formation occur in thrombo-embolism, arterial disease, and intravascular coagulation. Selective involvement in certain disease entities and combined involvement in others has been suggested on the basis of turnover studies. The development in this laboratory of sensitive and specific radioimmunoassays for two released platelet proteins, PF4 and βTG, and the availability of the radioimmunoassay for FPA as an index of fibrin formation have allowed studies of the physiologic basis for differential involvement of platelets and fibrin formation. Simultaneous measurement of platelet activation, monitored by radioimmunoassay for PF4 and βTG as well as aggregometry and 14C-serotonin (5HT) release, and FPA cleavage were carried out in citrated platelet rich plasma, whole blood and gel-filtered platelets. Collagen and ADP aggregated platelets and released 5HT, PF4 and βTG without detectable FPA cleavage indicating that thrombin action on fibrinogen is not involved in aggregation or release induced by these agents. Thrombin cleaved FPA at concentrations 100-fold less than those required for platelet protein release, and platelet protein release could be detected at lower thrombin concentrations than 5HT release. This might be due to greater sensitivity of the PF4 and βTG assays in detecting release or to different mechanisms of release of the proteins and 5HT. These results suggest that, in clinical samples, elevated FPA with normal PF4 and βTG might be due to concentrations of circulating thrombin sufficient to cleave FPA but too low to induce platelet release, and that the converse situation, with elevated PF4 and βTG but normal FPA might imply platelet activation by exposed subendothelial collagen with no thrombin action.
The Lysis of Early Fibrin by Eosinophils
Authors: Kwaan Hau C. , Hatem Ali A.
Published: May 2019

This study examins the role of leukocytes within a thrombus by demonstrating the morphologic detail of their activities, the chemotactic properties of thrombi and the presence of plasminogen and possible plasminogen activator within eosinophils. A model which produces discrete, reproducible platelet thrombi in arteries and veins of dogs allowed timed studies of their early evolution. In this model, the growth of the thrombus was constantly monitored by a flowmeter and the thrombus could thus be removed at a selected period in its formation. It was then studied histologically for fibrin activity and also ultrastructually. Little fibrinolytic activity was found. In contrast to neutrophils which are concerned particularly with the phagocytosis and disruption of platelet aggregates, we observed that eosinophils participate in the lysis and disruption of the fibrin within these aggregates. The fibrin is rarely phagocytosed but is acted on at the surfaces of the eosinophils, usually in shallow invaginations of the cell membranes. The fibrin shows morphologic changes of lysis. It appears that eosinophils and neutrophils are concerned with the transformation of the early fibrin and platelet thrombus, rather than with the resolution of the formed, mainly fibrin and red cell thrombus.
Disseminated Intravascular Coagulation and Irradiation
Authors: Wronowski T. , Tyrankiewicz Z. , Muszkowska J. , Kopeć M.
Published: May 2019

Studies of a group of 20 women treated for uterine cervix carcinoma with radium and 60Co revealed that in about two-thirds of patients fibrinogen degradation products /FDP/ appeared in urine and considerably increased in serum after the therapy. Under the conditions of the treatment kidneys were exposed to only negligible doses of radiation. In order to elucidate the observed phenomenon further, more detailed studies were performed on dogs. Four doses of 600 R of X-rays were given at two-days intervals either to the anterior or posterior part of the body. Irradiation resulted in a pronounced increase of serum FDP and appearance of FDP in urine which occured after the second dose. The highest values were gradually attained during the overt postirradiation sickness. These changes appeared independently of whether the front or back part of the body was shielded. The character and dynamics of changes were similar also in cases of fractionated local irradiation of either upper thorax or kidney region. Again there was no significant difference between the two groups irrespectively of the region exposed. Results of other coagulation tests and histological examination indicate that irradiation induced systemic activation of blood coagulation. Kidneys appear to be involved in this process in an indirect way.
A New Physiological Inhibitor of Blood Coagulation in the Newborn
Authors: Hemker H. C. , Muller A. D. , Doorm J. M. V.
Published: May 2019

In more than half of a series of samples of cord blood a hitherto unrecognized inhibitor of blood coagulation could be demonstrated. At three days of age this inhibitor is found in all babies. In 27 out of 43 samples the disappearance rate of thrombin in the two-stage prothrombin assay was significant (> 5 times) greater than it uses to be in normal human adult plasma. In the 16 others it was no more than 1.8 times greater than normal. On basis of this observation we divided the plasma in an I (inactivation) and non-I group. We determined thrombin inactivating proteins in I and in non-I plasmas respectively and found no significant differences in antithrombin 3, (α1-antitrypsin, α2-macroglobulin, antiplasmin, C1 esterase inhibitor or inter-α-antitrypsin inhibitor. A non—protein fraction can be prepared from blood containing this inhibitor that has no appreciable inhibitory action of its own but that markedly enhances the activity of antithrombin 3. We found as yet no reasons to assume that the inhibitor is something else than heparin.
Human Basement Membrane Collagens do not Elicit Platelet Aggregation
Authors: Trelstad R. L. , Carvalho A. C.
Published: May 2019

The immediate subendothelial connective tissue matrix consists of the basement membrane, a collagenous felt-like cell surface coat. The collagen from basement membranes has been isolated from human lung, skin, and kidney using a new fractionation method which separates native forms of collagen Types I, II, III, and IV. The Type IV collagens from the basement membranes have been characterized in respect to amino acid and carbohydrate composition, molecular size, charge and native structure. Antibodies prepared against the Type IV collagen reacted with both epithelial and vascular basement membranes as judged by immunofluorescence. Platelet-rich plasma (250,000/μl) from 5 normal subjects were tested for aggregation and 14C-serotonin release with human collagen Types I, II, III, and IV. Complete aggregation (100%) and 14C-serotonin release (80–100%) was obtained when Types I, II, and III were used. Human kidney, lung, and skin collagen Type IV (10–100μg/ml) did not aggregate platelets nor cause release of their contents. Pre-incubation of platelets and human collagen Type IV for periods of 30 minutes did not result in inhibition of platelet aggregation by Types I, II, or III.

These data indicate that the collagenous component of the basement membrane, the first extra-vascular collagen to which a platelet is exposed, does not elicit aggregation as do the fibrillar collagens in the perivascular matrix.
Studies on the Excretion of 3H-Heparin
Authors: Losito R. , Lemieux E. , Longpré B.
Published: May 2019

Disappearance of heparin is still not well understood;because of this, we studied the excretion of 3H-heparin in (a) rats with biliary fistula and cystotomy, (b) rats with cystotomy, (c) hepatectomized rats and (d) in the isolated perfused rat liver. During 5 hours of perfusion, there was never more than 8% of the % excreted into the bile;most of the radioactivity (80%) remained in the blood. In bile, one radioactive component, which possessed slight anticoagulant and metachromatic activity was found and appears to be similar to heparin on chromatography and microelectrophoresis. In the intact animal, utilizing similar doses, the biliary excretion was similar to that of the isolated system;in contrast to the isolated system, radioactivity was quickly removed from the blood (over 90% in 5 h) and excreted into the urine (30% and 60% after 5 h and 10 h respectively). In hepatectomized animals, it was observed that only 15% of heparin radioactivity could be excreted in 24 hours. Hence it is postulated that the liver appears to have an important role to plav in the metabolism and biological actions of heparin since (1) it removes 3n_heparin from blood and excretes it into the bile and (2) the results of the hepatectomized experiments showing that only one-fifth the heparin was excreted compared to the control rats without hepatectomy.
Heterozygosity and Homozygosity in von Willebrand’s Disease : A Study of 108 Cases
Authors: Shoa’i I. , Lavergne J. -M. , Ardaillou N. , Ala F. , Meyer D.
Published: May 2019

Factor VIII/Willebrand Factor (F. VIII/WF) resides on a multimeric glycoprotein under autosomal control. Von Willebrand’s disease (vWd) may be related to a quantitative (inability to produce F. VIII/WF) or to a qualitative defect (production of an abnormal F. VIII/WF). The study of 108 cases illustrates the various genetic defects. In 33 cases from 21 families, the severity of the disease was consistent with a homozygous state. All showed a total lack of Factor VIII related antigen (VIIIR:AG) by immunoradiómetry, contrasting with low (1–5%) but detectable Factor VIII procoagulant activity (VIII:C). 4 of these patients developed antibodies which precipitated VIIIR:AG and neutralized Willebrand Factor activity (VIIIR:WF). In 16 of these 21 families, the parents were first cousins and 84% of the 48 relatives studied showed an abnormally high ratio of VIIIrC to VIIIR:WF -together with a qualitatively normal protein- consistent with a heterozygous state. A further 8 unrelated patients showed a less severe defect (5–20% of VIII:C, VIIIR:AG and VIIIR:WF) with a qualitatively normal protein. The remaining 19 patients had higher levels of VIIIR:AG than of VIIIR:WF and a qualitatively abnormal protein as demonstrated by electrophoretic mobility, agarose elution pattern, precipitation by concanavalin A and dose-response curve by immunoradiometry.
Electron Microscopic Studies of the Factor VIII-Von Willebrand Factor Protein
Authors: Marder V. J. , Tranqui-Pouit L. , Hudry-Clergeon G. , Atichartakarn V. , Suscillon M.
Published: May 2019

Preparations of human Factor VIII which were homogemeous by SDS-polyacrylamide gel electrophoresis were examined by electron microscopy after negative staining with phosphotungstic acid. Most striking were large oval and circular forms, usually present in loose groupings of four or more molecules. Approximately 80% were oval-shaped and the mean axial dimensions of 167 such molecules, considered to be prolate ellipsoids, were 725 Å and 346 Å (axial ratio about 2rl). The 34 circular forms had a mean diameter of 545 Å and were considered to represent a separate though related molecular entity, either of spherical or oblate ellipsoidal shape. A variety of smaller forms was also present in these preparations including round molecules of 110–330 Å in diameter, irregular ovoid shapes with filamentous transformation and filaments of variable length and thickness. Based on calculations of volume relative to that of fibrinogen, the molecular weight of the predominant prolate ellipsoid form is about 3 million daltons, a value which is consistent with reported estimates of molecular size obtained by ultracentrifuge or gel filtration studies.
New Method for Aggregometry
Authors: Sano T. , Motomiya T. , Yamazaki H.
Published: May 2019

Platelet sensitivity to aggregation seemsto reflect some different aspect from other parameters of platelet function. A new method to assess platelet sensitivity to ADP-aggregation was devised. It has three advantages: it requires 1) no centrifugation, 2) small amount of blood and 3) inexpensive equipments. Venous blood (0.8 ml) was drawn into a polyethylene syringe containing 0.2 ml of citrate. PRP was sgparated by standing the syringe on a clay board and was added to ADP, serially diluted from 2-5 to 2-15mg/ml in a Microtiter tray. The minimum effective ADP concentration to give aggregation (2-nmg/ml) was detected microscopically and platelet sensitivity was expressed by the absolute value of the exponent (n) of the endpoint ADP concentration. Sensitivity correlated with intensity of aggregation obtained by the optical density (OD) and the screen filtration pressure (SFP) method, but not with velocity of aggregation. Using this method, thefollowing findings were observed. 1)Sensitivity was enhanced i)in acute stage of thrombosis, ii)after isometric excersise in the patients with coronary and cerebral arteriosclerosis and diabetic patients. 2)Sensitivity, but not intensity of aggregation, correlated with age (r= 0.66, P<0.005) in healthy males. 3)When human citrated blood was filtrated through glass filter, sensitivity was enhanced, while intensity of aggregation did not change. The degree of the enhancement of sensitivity increased as the contact area of glass increased. These findings suggest that the appearance of hypersensitive platelets may be reflected more on sensitivity than on intensity of platelet aggregation. This method is also applicable to ristocetin-induced platelet aggregation and von Willebrand factor assay.
Heterogeneity of Factor IX in Therapeutic Concentrates
Authors: Ménaché D. , Aronson D.
Published: May 2019

In vivo and in vitro experience shows that factor IX concentrates are partially activated. A rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”.

“Rocket” Immunoelectrophoresis of all concentrates showed a 1.5–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed Immunoelectrophoresis of standard factor IX preparation produced a single sharp peak whether electrophoresed in Ca or EDTA containing buffer. “Activated” preparations produced a dome shaped precipitin arc. The addition of plasma to factor IX concentrates yielded a marked shoulder only when the electrophoresis was done in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to the activated IX (IXa) revealed an even more pronounced heterogeneity whether in Ca or EDTA. The addition of AT-III produced a second precipitin peak when activated IX was electrophoresed in the presence of Ca++.

These results indicate that at least three forms of factor IX exist in factor IX preparations. The absence of detectable AT-III reacting material in the regular IX preparations is a priori evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” complex is evidence of biologically active material.
Factor Xa Like Enzyme in Snake “Habu” Venom
Authors: Maruyama I. , Kinoshita T. , Kazama M. , Igata A. , Abe T.
Published: May 2019

The snake “HABU” venom was divided into subfractions and their influence on both platelet and coagulation were studied. Four fractions were obtained by gel exclusion chromatography on Sephadex G75 of which molecular weight ranged from 100,000 to 5,000. The fraction of the largest molecular weight was further divided into four subfractions; I–1, I–2, I–3 and I–4 by DEAE cellulose chromatography. The subfraction I–1 showed no activity to induce platelet aggregation but suppressed ADP, collagen and ristocetin-induced aggregation. The subfraction I–2 and I–3 equally induced the aggregation of plasma platelet and gel filtrated platelet suspension. The subfraction I–4 had the same effect on the platelet aggregation except for the specific suppressive effect on the restocetin aggregation. The subfraction I–3 was found to have Xa like activity which was revealed using chromogenic substance (S–2222). This activity was not inhibited by purified human antithrombin III, α2-macroglobulin or α1-antitrypsin. The relationship between activites of these fractions on platelet and coagulation suggested that they are different from any known coagulation factor.

HABU--- Trimeresurus flavoviridis Hallowell
Fibrinopeptide B and Aggregation of Fibrinogen
Authors: Shainoff John R. , Dardik Beatriz N.
Published: May 2019

Copperhead venom procoagulant enzyme, previously shown to remove fibrinopeptide B at faster rate than fibrinopeptide A, has been found to release little of the A peptide at temperatures below 14°. At low temperatures, tight aggregation of the derivative lacking B blocked release of A by the enzyme. Transient release of A occurred, but stopped as removal of B approached completion. Overall losses of A amounted to 8% and 2% from human and rabbit fibrinogen respectively. Resultant clots with A intact dissolved on warming to 37°, whereafter release of A resumed with secondary coagulation ensuing. When dissolved at 37° with PMSF-inactivated enzyme, the fibrin remained highly soluble (>12 mg/ml). Ultracentrifugation showed constant levels of 8S monomer together with 16S aggregates in amounts accounting for the total protein at concentrations down to 0.16 mg/ml, and monomer alone at lower concentrations. From changes in saturation level of monomer at lower temperatures, the enthalpy of aggregation appeared to be about half of the -50 kcal/mole associated with aggregation of regular fibrin. Other experiments indicate that removal of the A and B peptides unmasks separate aggregation sites, each of which place the monomers in alignment suitable for rapid crosslinking. The β-chain segment spanning residues 15-42 appears to be critically involved in the aggregation that follows release of B itself, because the aggregation does not occur when this segment is removed together with B in early stages of reaction between fibrinogen and plasmin. The aggregation which follows release of A depends on a different site, because the early alterations inflicted by plasmin do not prevent coagulation of fibrinogen by thrombin. (Supported in part by USPHS Grant HL-16361).
Acquired Dysfibrinogenemia in Acute and Chronic Liver Disease
Authors: Lane D.A. , Kakkar V.V. , Woolf I.L. , Williams R.
Published: May 2019

Recent work with the thrombin-like snake venom Reptilase has suggested that the incidence of dysfibrinogenemia in liver disease may be much higher than has been recognised previously. In the present study, nine patients with fulminant hepatic failure and three with chronic liver disease, all exhibiting prolonged thrombin and ancrod clotting times, were examined for evidence of dysfibrinogenemia with a variety of physiochemical and clotting techniques. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis revealed normal fibrinogen polypeptide chains, polypeptide chain crosslinking by Factor XIII, and in vitro generated plasmin degradation products of fibrinogen (FDP) in the plasma of these patients. Isolated and purified fibrin monomers were prepared by clotting plasma with Reptilase (Reptilase monomer) and thrombin (thrombin monomers). This enabled fibrin monomer polymerisation to be studied free of interference from plasma inhibitors such as FDP, and independent of any abnormal fibrinopeptide release. Delayed polymerisation of both Reptilase and thrombin monomers was demonstrated, providing an explanation for the observed prolonged clotting times. These results confirm that an abnormal fibrinogen may be produced during acute and chronic liver disease.
Isolation of Some Platelet Membrane Glycoproteins
Authors: Clemetson K.J. , Pfueller S.L. , Sturk A. , Lüscher E.F. , Jenkins C.S.P.
Published: May 2019

The platelet is surrounded by a pronounced glycocalix formed by carbohydrate moieties of the membrane glycoproteins. The number of glycoproteins of the outer platelet membrane is greater in number than had previously been reported : when solubilized membranes are analyzed by SDS-polyacrylamide gel electrophoresis the number of separated carbohydrate entities was found not only to be dependant on the concentration of acrylamide and of bisacrylamide used but also on the buffer system employed.

The major platelet membrane glycoproteins have been solubilized and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. SDS-polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven glycoprotein entities. Using combinations of lectin columns, two platelet membrane glycoproteins have been isolated and others have been greatly purified.
Identification of the Thrombin-Binding Portion of Platelet Glycocalicin
Authors: Jamieson G.A. , Okumura T.
Published: May 2019

Glycocalicin is a high molecular weight (Mr 150,000) glycoprotein of the platelet glycocalyx which is obtained in soluble form following platelet homogenization. Under carefully controlled conditions, glycocalicin can be cleaved by endogenous or exogenous proteases (trypsin 1: 100, 2 min) to yield a macroglycopeptide (Mr 120,000) and a (nonglyco)peptide (Mr 45,000). These two fragments and intact glycocalicin have been compared with respect to their ability to prolong the thrombin clotting time of fibrinogen and to inhibit the binding of thrombin to intact platelets. The platelet macroglycopeptide was without effect in either system. The clotting time of fibrinogen (2mg/ml) by thrombin (2 u/ml) was prolonged from a control value of 16 sec to a value of 28 sec with glycocalicin (45 ug) and to 46 sec with the (nonglyco) peptide (6.5 ug). The binding of thrombin to intact platelets, as assayed by the Majerus technique, showed competitive inhibition with both glycocalicin and the (nonglyco)peptide, similar Ki values of 20-30 nanomolar being obtained in each case. These results indicate that the thrombin-binding site of platelet glycocalicin is located in the (nonglyco) peptide “tail” portion of the molecule.
Isolation of Human Antithrombin-III by Affinity Chromatography on Heparin-Agarose
Authors: Holleman W.H. , Coen L.J. , Capobianco J.O. , Barlow G.H.
Published: May 2019

Methods were developed for the isolation of gm. quantities of human antithrombin-III (AT-III) from Cohn Fraction IV-1 of human plasma using heparin covalently attached to agarose. Attachment of heparin carboxyl groups to alkylamino-agarose yielded a support with no affinity for AT-III. Linkage via the heparin hydroxyl groups yielded a support with approximately 1 mg of heparin/ml agarose and with a low capacity for binding AT-III. Linkage of heparin to agarose thru its amino groups yielded a heparin-agarose with the highest capacity for AT-III. Reaction of heparin containing a free α-amino group with cyanogen bromide activated agarose resulted in agarose substituted with 5 mg of heparin/ml. The conditions of buffer, pH, ionic strength and temperature which maximized AT-III binding were 0.05M sodium phosphate, 0.02M sodium citrate, 0.15M NaCl, pH 8.3 and 4°. The heparin-agarose bound 0.1-0.2 mg of AT-IIl/ml. The AT-III isolated by affinity chromatography was further purified by gel permeation and yielded a homogeneous product as judged by Polyacrylamide disc gel electrophoresis of native and reduced protein and by sedimentation velocity (S20, w = 4.1). This material had an activity of 1700 units/A280 as measured by inhibition of human thrombin. The AT-III is stable to heating at 60° for 10 hours in a buffer of 0.5M sodium citrate at pH 7-8. Injection of bovine thrombin (3000 units/Kg) into heparinized dogs (150 units/Kg) decreased circulating AT-III levels to 50%. (Supported by NHLI, Contract NOl-HB-4-2946).
Synthetic Polymers are Made Non Thrombogenic by Stable Coupling with an Antiplatelet Agent
Authors: Mari D. , Cattaneo M. , Gattinoni G.A. , Dioguardi N.
Published: May 2019

A number of porous fibres made of synthetic polymers (cellulosetriacetate) entrap enymatic systems without altering their biological activity. Such fibres inserted in extracorporeal circuits have a potential clinical applicability to remove toxic substances involved in the pathogenesis of acute liver failure. Since platelets (p1) adhere extensively when fibres are exposed in vitro to human blood, the possibility was evaluated to affect thrombogenicity by coating the fibres with antithrombotic substances. A preliminary screening carried out in vitro by means of a model of p1 adhesiveness to plastic columns filled with fibres showed that the antiplatelet agent ditazol (D) was the most effective compound in reducing p1 adhesion. D was further evaluated with more sensitive experimental models involving 51Cr labelled pl. Fibres were wrapped around a bob rotating in blood for different periods. P1 adhered massively within the first minutes to the uncoated fibres whereas both the rate and extent of adhesion decreased when fibres were coated with D. Heparinized blood flowing in a circuit by means of a pump insuring a constant flow rate of 500 ml/min was filtered through a cartridge inserted in the circuit and filled with coated fibres. Adhesion of 51Cr p1 to coated fibres was significantly lower than to uncoated fibres. Scanning EM showed scattered individual p1 adhering to coated fibres whereas large aggregates formed on the uncoated fibres. These findings show that stable coupling of an antiplatelet agent to synthetic polymers markedly reduce their thrombogenicity.