bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombin-Induced Changes in Phosphatidic ACID (PA) and Phosphoinositides
Authors: Leung N. L. , Kinlough-Rathbone R. L. , Mustard J. F.
Published: May 2019

Thrombin-induced changes in PA, monophosphoinositide (MPI) , diphosphoinositide (DPI) and triphosphoinositide (TPI) of washed rabbit platelets have been examined. Platelets prelabeled by incubation with 32P-orthophosphate, 3H-glycerol, 3H-inositol or 14C-arachidonate were exposed to 0.33 u/ml thrombin for one minute, and the phospholipids extracted and separated by thin layer chromatography. Measurement of absolute amounts of PA and MPI by phosphorus assay showed that PA increased by 180% while MPI decreased by 15%.

The changes in MPI with 3H-G, 3H-I and 14C-AA and in 1,2-diacyl glycerol (DG) indicate that some MPI may be converted to PA via DG. Changes in the 3H-G and 3H-I labeling of DPI and TPI suggest that the changes observed with 32P-Iabeled platelets are a result of the turnover of the phosphorylinositol moiety. The increase in AA with 14C-AA indicates that some of the decrease in MPI may be due to the formation of lyso MPI and free fatty acid. These results indicate that thrombin stimulation of platelets may affect inositol phospholipid metabolism through three pathways: (1) involving PA, DG and MPI; (2) the cleavage of free fatty acids from MPI; (3) turnover of the ester phosphates on DPI and TPI.
Functional Characterization of Membrane Sialyltransferase Activity of Human Platelets
Authors: Wu K. K. , Ku C. , Smith C.
Published: May 2019

To evaluate the role of membrane sialyltransferase in the initiation of platelet aggregation, we studied the stimulatory effect of epinephrine and adenosine diphosphate and the inhibitory effect of aspirin on the platelet surface sialyl transferase activity. The enzyme activity was assayed under optimal conditions as determined previously. The assay mixture consisted of intact washed human platelets, CMP-14C-sialic acid, desialated fetuin, Mn2+ and buffer to a final volume of 1 ml. The enzyme activity was enhanced to 172% of control by 1μH, 152% by 5μM and 146% by 10μM epinephrine. Adenosine diphosphate enhanced the enzyme activity to a lesser extent:103% at 1μM and 113% at 5μM. In contrast, aspirin inhibited the enzyme activity to 46% of control when 10μg/ml of aspirin was used. Higher concentrations of aspirin failed to cause further inhibition. In the in-vivo experiment, 600 mg aspirin was given to normal subjects and the surface enzyme activity was determined 12 hours later. The enzyme activity reduced to 43% following aspirin administration. Furthermore, we studied the enzyme activity in a patient with “aspirin-like” release disorder. While the mean surface enzyme activity of 10 normal subjects was 1.56 + 0.21 (S. D.) pmole-hr-1 per 108 platelets, the enzyme activity of the patient was only 0.91 pmole.hr-l. The results strongly suggest that the membrane sialyltransferase plays an important part in the initiation of platelet release reaction.
The Modification of Factor VIII Procoagulant Activity in Tissue Culture Media Supplemented with Fetal Calf Serum
Authors: Stead Nancy W. , McKee P. A.
Published: May 2019

Cultured human umbilical vein endothelial cells produce a protein that reacts with rabbit antibody to purified plasma factor VIII (FVIII)/von Willebrand factor (vWF) and has vWF activity, but not FVIII procoagulant activity (PCA) (Jaffe et al. JCI 52: 2757, 1973) . In our laboratory endothelial cell cultures produce vWF activity that increases to 3 µg/culture by 72 hrs; partially purified protein from culture media forms an immunoprecipitin line of identity with human, but not bovine, plasma FVIII/vWF. Of the 3 functions of plasma FVIII/vWF protein, PCA, but not vWF activity or antigenicity, is modified by trace proteases. An assay that measures hydrolysis of p-nitroaniline from the tripeptide Bz-phe-val-arg-p-nitroanilide detects trace proteases in growth media; 0.001 NIH u thrombin releases measurable quantities of p-nitroaniline. Culture media without fetal calf serum (FCS) did not hydrolyze the tripeptide. Media 20% in: FCS, FCS heated to 56° for 30 min, FCS made 1.7×10-3M diisopropylfluorphosphate (DFP), FCS and 4.4x10-8 moles soybean trypsin inhibitor, or FCS and 4.4xl0-8 moles Trasylol releases 3.5x10-6, 3.6x10-7, 3.6xl0-7, 3.5xl0-6 and 2.8xl0-6 mmole of p-nitroaniline per ml of media per min. PCA of FVIII/vWF molecule is measured by one stage (PCA1) and two stage (PCA2) assays. Although >85% PCA1 or PCA2 of purified plasma FVIIl/vWF added to media without FCS remains at 6 hours, varying amounts of PCA1 and only 10% PCA2 of the FVIII/vWF added to media 20% in FCS remained in 6 hrs. Hence endothelial cells make a protein with vWF activity and FVIII/vWF antigenicity; but were the protein to have PCA at the time of release, trace proteases in the growth media could quickly destroy it.
Stabilization of the Antihemophilic Factor (VIII :AHF) by the Von Willebrand Factor (VIII :VWF): A Possible Model for Von Willebrand’s Disease
Authors: Weiss H. J. , Sussman I. I. , Hoyer L. W.
Published: May 2019

When compared with VIII:AHF in normal citrated plasmas, VIII:AHF activity showed increased lability at 37°C in the ‘late’ post-transfusion plasmas (VIII:AHF≫VIII:VWF) of a patient with von Willebrand’s disease, but not in the ‘early’ post-transfusion plasmas in which VIII:AHF~VIII:VWF. VIII:AHF was also labile in the baseline plasmas of 3 patients with von Willebrand’s disease in whom VIII:AHF≫VIII:VWF. In two of these patients the mobility of Factor VIII antigen (on crossed Immunoelectrophoresis) was increased. (VIII:AHF was not excessively labile in 4 other patients in whom VIII :AHF~VIII:VWF). In all of the above cases, VIII:AHF was stabilized by the addition of either purified von Willebrand factor or plasmas of patients with hemophilia, but not by plasmas of patients with severe von Willebrand’s disease. Thus, VIII:VWF may serve to stabilize VIII:AHF and this might explain the post-transfusion findings in von Willebrand’s disease.
Relation of Growth Hormone and Von Willebrand Factor Activity
Authors: Sarji K. E. , Schraibman H. B. , Levine J. H. , Barnes M. , Nair R. M. G. , Sagel J. , Colwell J. A.
Published: May 2019

Growth hormone (GH) has been implicated in the pathogenesis of diabetic angiopathy although the exact mechanism remains unknown, von Willebrand factor activity (vWF) is significantly higher in diabetic than in normal plasma (p<.001). In view of this, we have investigated the relationship of radioimmunoassayable GH and vWF activity, as measured in a bioassay using washed normal human platelets and ristocetin. Samples were obtained after an overnight fast, during oral glucose tolerance tests, and during sleep. A significant positive correlation was seen between GH over a range of 0.46 to 12.2 ng/ml and vWF activity over a range of 1 to 470% (p<.01). vWF activity was suppressed by oral glucose in normals and diabetics, with the amount of suppression related to the degree of glucose intolerance. Maximal suppression of vWF activity was coincident with maximal GH suppression. Samples from 4 normal subjects drawn through an indwelling catheter at 30-minute intervals during sleep showed peaks of plasma GH and vWF activity with GH peaks preceding vWF peaks by an hour or less. Two patients with panhypopituitarism and one patient with isolated GH deficiency were similarly studied. GH levels were low in these patients, but vWF levels were normal; both GH and vWF showed little sleep-related change.

A single intramuscular injection of GH produced a marked increase of vWF activity in all 3 patients within 30 minutes and a later increase after 5–6 hours. We conclude that a regulator of glucose metabolism, possibly GH, is involved in regulation of vWF activity.
Activation of Hageman Factor (HF, Factor XII) by Sephadex-Ellagic Acid Mixtures
Authors: Ratnoff O. D. , Saito H.
Published: May 2019

In the intrinsic pathway of clotting, PTA (Factor XI) is activated by activated HF. Whether activation of HF requires the presence of prekallikrein (Fletcher factor) and high M. W.(HMW) kininogen (Fitzgerald factor), or whether these agents act after activation of HF is disputed. Purified HF activates PTA in the absence of prekallikrein, but this may reflect molecular changes in HF during purification.

Sephadex G10 or G15 was mixed with ellagic acid and freed of excess ellagic acid by washing. A mixture of normal plasma, or plasma deficient in HF, prekallikrein, HMW kininogen or PTA, and Sephadex-ellagic acid was centrifuged at 31000 xg. Each supernatant plasma except that deficient in HF shortened the abnormally long partial thromboplastin time (PTT) of HF-deficient plasma. Plasma simultaneously depleted of HMW kininogen, prekallikrein and PTA also shortened the PTT of HF-deficient plasma and of plasma depleted of HF and prekallikrein (albeit minimal clotting times were reached more slowly in the latter case) but had virtually no procoagulant effect upon the PTT of plasma depleted of HF and HMW kin inogen.

Thus, exposure of HF in plasma to Sephadex-ellagic acid generated a clot-promoting form of HF in the absence of other clotting factors, but its full expression requited the presence of HMW kininogen.
Rapid Loss of Factor XII- and XI-Activity in Ellagic Acid Activated Normal Plasma: Role of Plasma Inhibitors and Implications for Automated Aptt-Recording
Authors: Joist J. H. , Cowan J. F. , Khan M.
Published: May 2019

Rapid prolongation of the activated partial thromboplastin time (aPTT) of normal plasma upon incubation with ellagic acid containing aPTT reagents was observed. The aPTT prolongation was not due to time dependent changes in pH in the incubation mixtures or loss of activity of the labile coagulation factors VIII and V, but occurred as a result of rapid progressive inactivation of ellagic acid activated factors XII and XI. Prolongation of the aPTT and loss of contact factor activities was not observed in plasma incubated with particulate activator reagents. This finding seemed to indicate that adsorption of factors XII and XI to larger particles during the activation process may protect these factors from inactivation by naturally occurring plasma inhibitors. Evidence is presented which supports previous observations that Ci-inhibitor, α1-antitrypsin and antithrombin III (in the presence of heparin contribute to factor XIIa- and XIa-inactivation in ellagic acid activated plasma and that plasma albumin may compete with factor XII for ellagic acid binding. The findings indicate that ellagic acid containing aPTT reagents have unfavorable properties which seriously limit their usefulness in the clinical laboratory, particularly in respect to recording of the aPTT with certain fully automated clot timers.
Thrombin Generation Through the Short-Circuit XII–XI–VII
Authors: Altman R. , Coiodner C.
Published: May 2019

Altman and Hemker reported the activation of factor VII by factors XII and XI and it seems to us important to demonstrate the possibility of thrombin formation through this short-circuit between the intrinsic and extrinsic clotting system. Kaolin particles coated with human fibrinogen clumps by effect of thrombin. With concentrations of 0.1U/ml or more, clumping was clearly visible. Hirudin prevent the clumping of the kaolin coated particles. When kaolin was incubated with citrated plasma, we supposed that fibrinogen is adsorbed on kaolin surface. Then, various deficient plasmas were tested. In plasmas deficient of factor XII, XI or II, none clumping were obtained. In factor VII deficient plasma, little degree of effect was observed. The addition of contact product (CP) to Hageman plasma returned its clumping capacity. This effect is not due to CP itself. Ca++ seems to be necessary for this action because EDTA 3mEq, inhibit the clumping capacity of normal plasma. We concluded that factors XII, XI, VII, II and Ca++ are absolutely necessary for the thrombin generation in these experiments, whereas factors IX, VIII, X and V are not.
Studies on Fractionated Heparin
Authors: Lane P. A. , Macgregor I. , Michalski R. , Kakkar V. V.
Published: May 2019

Commercial porcine heparin has been divided into five different molecular weight components following gel filtration upon a polyacrylamide-agarose gel matrix. Rechromatography has shown that the two extreme fractions, which contain very low and very high molecular weight material, were totally separable upon gel filtration, while the intermediate fractions contained material in common with other fractions. Four of the five fractions contained almost equivalent specific activity when measured by an anti-Factor Xa clotting assay (l) and yet had very different specific activities if assayed by either thrombin clotting time or kaolin-cephalin clotting time methods. The highest MW fraction had low specific activity in all of the clotting assays, suggesting that it contained the greatest percentage of a non-reactive material demonstrated by Rosenberg et al, (2) while the lowest MW fraction had low activity only in thrombin clotting time and KCCT assays. The lowest MW fraction produced a smaller acceleration of purified fibrin monomer polymerisation rate and also smaller inhibition of thrombin induced platelet aggregation in plasma. The demonstration of different results for specific activities of the heparin fractions, depending upon the assay method used is in agreement with a recent study (3) and has important implications in regard to the pharmacopeal assays for heparin. It also suggests that heparins with higher specific anti-Factor Xa (or antithrombotic) effect can be prepared from commercial heparins, although these may have a relatively low ability to neutralise thrombin in plasma.
Anticoagulant Activities of Lung and Mucosal Heparin
Authors: Barrowcliffe T. W. , Johnson E. A. , Eggleton C. A. , Thomas D. P.
Published: May 2019

Interaction with antithrombin III is thought to be the main mechanism whereby heparin exerts an anticoagulant effect, but measurements of this specific heparin activity by an anti-Xa assay do not always agree with measurements made by ‘multiple role’ assays, such as APTT or pharmacopoeial assays. Two batches of lung heparin had APTT activities in vitro about 1.4 times those found by anti-Xa, whereas in several batches of porcine mucosal heparin this ratio was about 0.8. All assays by both methods were carried out against the 3rd International Standard for heparin. After gel filtration, lung heparin maintained a high ratio of APTT to anti-Xa activity in all except the low molecular weight fractions, where the two activities were both about 60 i. u./mg. In contrast, low molecular weight mucosal fractions had negligible APTT activity, but high (120 i. u./mg) activity by anti-Xa assay. A nominal 1000 units of lung heparin injected I. V. into volunteers gave peak anti-Xa levels of about 0.2 i. u./ml; a comparable injection of mucosal heparin gave peak levels of about 0.3 i. u./ml. The resulting ratio agreed with the anti-Xa activities of these two batches in vitro. However, in vivo, APTT levels with both heparins were less than half the anti-Xa levels, and 50 mins. after injection there was virtually no effect on the APTT, while heparin levels by anti-Xa remained about 0.1 i. u./ml. Although their APTT activities were comparable, lung heparin had much less anti-Xa potentiating effect than mucosal heparin, both in vitro and in vivo; this has important implications for the assay and clinical use of heparin.
Effects of Pyrazolone Drugs on Synthesis of Prostaglandin D2 and Thromboxane B2 by Platelets
Authors: Ali M. , Zamecnik J. , McDonald J. W. D.
Published: May 2019

The principle products of arachidonic acid (AA) in platelets are hydroxylated fatty acids and thromboxane B2(TXB2). Prostaglandin D2(PGD2) has been considered to be a nonenzymatic degradation product of prostaglandin H2 formed in the presence of plasma albumin. Using 14C AA as substrate and thin layer and silicic acid chromatography, we have demonstrated PGD2 synthesis by washed (albumin-free) human platelets. The identity of PGD2 was confirmed by gas chromatography-mass spectrometry. In platelets lysed by freezing and thawing synthesis of TXB2 and PGD2 was approximately equal and equally inhibited by pyrazolones.

Synthesis of PGD2 by platelets is enzymatic and may contribute to bronchoconstrictor, vasomotor, and inflammatory effects induced by platelet aggregation. Pyrazolones appear to inhibit cyclooxygenase activity rather than the breakdown of cyclic endoperoxides as previously postulated.
Independent Induction of Platelet Aggregation and Secretion by Prostaglandin Endoperoxide Analogues and Thromboxane A2-Like Material
Authors: Charo I. F. , Feinman R. D. , Detwiler T. C. , Smith J. B.
Published: May 2019

We have investigated the mechanims of platelet activation by two prostaglandin endoperoxide analogues (U-46619 and U-44069) using a new instrument that simultaneously monitors platelet aggregation and secretion. Low concentrations of these compounds induce platelet aggregation without secretion (i. e., primary aggregation), while slightly higher concentrations induce biphasic aggregation with secretion paralleling the second phase. At still higher concentrations, aggregation and secretion begin simultaneously, and in the absence of stirring there is secretion but no aggregation. A critical concentration of endoperoxide analogues can often be found that will induce 2 waves of secretion, a phenomenon not seen with other stimuli. Similar results were obtained with thromboxane A2-like material that was generated by incubation of dog platelets with Na-arachidonate. In contrast, when platelets are stimulated with Na-arachidonate, the precursor of the endoperoxides and thromboxanes, we never observe significant aggregation without secretion, and even at the lowest concentrations secretion is independent of aggregation. We conclude that both the prostaglandin endoperoxide analogues and thromboxane A2-like material induce platelet aggregation independent of released ADP and only at higher concentrations can directly induce secretion, whereas Na-arachidonate induces aggregation and secretion in parallel.
Fluorogenic Substrates for the Determination of Plasminogen Activators and Plasmin
Authors: Nieuwenhuizen W. , Wijngaards G. , Groeneveld E.
Published: May 2019

Plasminogen activator activities in biological fluids are low and specific for plasminogen. Highly sensitive and accurate assays are needed for direct quantification and for kinetic studies. We therefore, synthesized tripeptide amides i. e., t. BOC. val. gly. arg β-NA (l) and val. gly. arg β-NA (II) from which the fluorescent group β-naphtylamine (β-NA) is released upon enzymatic hydrolysis.

Kinetic parameters found for the hydrolysis of these substrates by plasmin, urokinase and tissue plasminogen activator are:

*in nmol/CU/min; **in pmol/CTA/min

Strikingly, tissue activator is not active on substrate II, allowing discrimination between this activator and other proteases.

The sensitivity of the assay is at least ten times higher than that observed for other synthetic substrates available. Moreover, substrates I and II can be used in enzyme histochemistry.
Adsorption of F VIII on Aluminium Hydroxide
Authors: Seghatchian M. J. , Barrowcliffe T. , Miller-Andersson M.
Published: May 2019

Adsorption of plasma by A1(OH)3 is a requirement for the two stage assay of F VIII. It is generally accepted that factors II, VII, IX and X are removed by the procedure, while factors V and VIII are unaffected. Following gel filtration of a F VIII concentrat on Sepharose 4 B F VIII:c was found in the low molecular weight area, as well as in the void volume as expected. This activity was found with both one and two stage techniques. After adsorption of the fractions with Al(OH)3 to eliminate the non F VIII procoagulant activity F VIII:c disappeared from the void volume fractions and was much reduced in the low molecular region. F VIII: R Ag was also removed from these fractions by A1(OH)3 adsorption. After adsorption of fractions in the presence of hemophilia plasma clotting activity remained in both regions suggesting the presence of true F VIII activity. Thus at concentration of 1 IU of F VIII:c per ml, a low purity preparation was unaffected by A1(OH)3 adsorption whereas both antigen and clotting activity of a high purity concentrate were conciderably reduced. Addition of 5 % albumin to the high purity preparation prevented this adsorption. It is concluded that under conditions of high purification F VIII:c can be adsorbed preferentially on A1(OH)3 and this appears to be due to removal of F VIII:R Ag.
Problems of Quantitation of F VIII:C Activity
Authors: Nilsson I. M. , Barrowcliffe T. , Kirkwood T. , Miller-Andersson M. , Seghatchian M. J.
Published: May 2019

The establishment in 1970 of the first international standard for F VIII by the expert committee on biological standardization of the WHO achieved a major step towards uniform measurement of F VIII:C activity. This has allowed different laboratories to make detailed comparisons of their assay systems. These studies, which include the recently completed international collaborative study of the proposed 2nd I S have repeatedly shown that systematic differences between laboratories still remain. In particular, two-stage assays are detecting relatively more activity, on average, in the more purified preparations than one-stage assays. The discrepancy between methods may be as high as 20–25 %. Also, even with the same methods, different sets of reagents are giving substantially different results. The fundamental characterization required of a F VIII preparation is a measure of its in vivo effect. Present understanding of F VIII biochemistry suggests that this effect may be the product of complex interactions between several different activities. Nevertheless the world price of F VIII preparations in determined largely in terms of units of clotting activity. The discrepancies between assay systems thus have significant financial as well as clinical and scientific implications. This suggests that international agreement should be sought as to which assay systems may be relied upon to give realistic measures of potential in vivo effect.
Immunological Characterisation of A∝ Chain Fragments of Human Fibrinogen
Authors: Conkie J. A. , Davidson J. F.
Published: May 2019

In an investigation of the early cleavage fragments resulting from fibrin(ogen)olysis, we have examined the nature of small peptides arising from the C-terminal part of the Aα chain of human fibrinogen.

Antiserum to the carboxymethylated Aα chain of human fibrinogen has been prepared, and used to study the Aα-related antigens from (i) a plasmin digest of human fibrinogen, (ii) human serum, obtained from urokinase-treated normal plasma, and (iii) normal human serum. Using immunodiffusion and immunoelectrophoresis with anti-Aα antiserum, along with Polyacrylamide gel electrophoresis, at least two non-identical Aα -related antigens have been detected in a final plasmin digest of human fibrinogen. The largest of these antigens (MW 26,000) has been isolated and named Aα.-RA (26,000). This may be similar to the previously-described fragments, Hi2-Ala, fragment A or fragment H. Aα -related antigen has been produced by in vitro fibrinogenolysis in plasma and has been detected in the serum. In addition, small quantities of Aα -related antigen have been found in untreated normal human serum.

These results suggest that anti-Aα antiserum is likely to be a useful reagent for the study of the early plasmin-derived fragments of fibrin(ogen)olysis in vitro and in vivo.
Localization and Characterization of the Cleavage-Associated Neoantigen in the Edomain of Fibrinogen
Authors: Plow E. F. , Edgington T. S.
Published: May 2019

Plasmic cleavage of fibrinogen to generate fragment X partially exposes a specific cryptic molecular site, fg-Eneo. This site in the E domain of the molecule is further exposed during subsequent cleavage. We now report on localization of this site which provides an incisive marker for the structural and conformational changes associated with plasmic cleavage of fibrinogen. Fg-Eneo was stable to reduction and alkylation and the chains of the E fragment were separated by ion exchange chromatography on DEAE-cellulose. An active component was obtained and subjected to molecular exclusion chromatography on Sephadex G-50 to insure removal of intact fg-E. A fg-Eneo positive chain was recovered and identified as Eγ with respect to amino-terminal tyrosine, amino acid composition, and immunochemical analysis. The fg-Eneo site was stable to tryptic degradation, and tryptic peptides were prepared and separated by multiple molecular exclusion chromatographic steps. Final separation of two peptides of similar size was achieved on the basis of carbohydrate content by affinity chromatography on Concanavalin A. Only the active peptide was bound by the lectin. Purity and identification of the active tryptic peptide as γ36–53 was established by amino acid composition and sequence. These results establish that this region of the γ chain of fibrinogen is not present at the hydrated surface of the native molecule but that, in association with plasmic cleavage and conformational changes, this site is progressively exposed and provides a dynamic marker of the cleavage sequence.
Malondialdehyde (MDA) Formation in Patients with Defective Release Reaction
Authors: Pareti F. I. , Mannucci L. , Capitanio A. , Mills D. C. B.
Published: May 2019

MDA formation in platelet rich plasma (PRP) is considered to be an indicator of prostaglandin endoperoxide production by the platelets, occurring during the release reaction. Six patients with a defect of the release reaction, storage pool deficiency (SPD), due to the lack of the storage pool of ADP, showed a normal amount of MDA formation in PRP after aggregation with collagen, thrombin (5U/ml) and after incubation with 10 mM N-ethyl-maleimide (NEM) . The addition to PRP of 10 μM ADP and 1 μM serotonin (5HT) , amounts comparable to those released during the release reaction, together with high concentrations of collagen, thrombin or NEM did not significantly enhance MDA formation. This suggests that MDA production is independent of released ADP and 5HT, and that the release induction mechanism is normal in these patients. Platelets from one patient with the “aspirin-like syndrome” did not produce MDA after stimulation with collagen, thrombin or NEM; neither did those from two normal donors after aspirin ingestion. The “aspirin-like syndrome” seems to be characterized by a specific defect in the release mechanism due to the inactivity of the cyclo-oxygenase, while the release mechanism is normal in SPD; the defect being due solely to the absence of releasable ADP.
Ultrastructural and Partial Biochemical Characterization of Platelets from Chediak-Higashi Cattle
Authors: Meyers K. M. , Seacord C. I. , Hopkins G. , Holmsen H.
Published: May 2019

To provide additional information on the platelet defect which is associated with the Chediak-Higashi syndrome (CHS), platelet rich plasma from normal and CHS cattle was incubated with 14C-adenine. Platelets were then isolated by gel filtration and treated with thrombin. Both the resting amount and extent of secretion of ATP, ADP, several acid hydrolasis, serotonin, calcium and magnesium was determined. Nucleotide profiles and electron micrographs of resting and thrombin treated platelets were also obtained. The markedly reduced secretion of nucleotides, serotonin, and metals demonstrate that CHS cattle have a storage pool defect. Furthermore, there appears to be significant differences in both the resting amount and extent of secretion of several of these measured substances between normal cattle and human platelets.
Incorporation of3H-Glycerol into Phospholipids of Normal and abnormal Platelets
Authors: Hutton R. A. , Hardisty R. M. , Deykin D.
Published: May 2019

Glycerol-2-3H uptake into phospholipids separated by unidirectional chromatography on silica gel H plates, was measured in normal platelets and those from patients with Glanzman’s thrombasthenia (GT), Bernard-Soulier syndrome (B-S), Storage pool deficiency (SPD) or aspirinlike release defect . The results were compared with the release of adenine nucleotides (AN) and with changes in platelet factor 3 availability (PF3a). In normal platelets, secondary aggregation induced by adenosine diphosphate (ADP), Epinephrine (EPI) , Collagen (COL) or Ristocetin (RI) led to a marked stimulation of glycerol uptake over a 60 minute period of incubation, the most striking increase being in the phosphatidyl inositol (PI) fraction.

No quantitative differences in phospholipid distribution were noted in any of the patients tested. In all cases, the uptake of glycerol was proportional to the degree of platelet aggregation, being minimal where no aggregation was observed (GT platelets mixed with ADP or EPI) , of intermediate level where primary aggregation alone occurred (SPD platelets with all agents or with GT platelets with RI), and similar to normal where secondary aggregation was induced (B-S platelets stirred with ADP or EPI). The results suggest that the defective 3H-glycerol uptake by abnormal platelets is a consequence rather than a cause of the decrease in platelet aggregation. Enhanced PI turnover most probably reflects the cells response to membrane damage or distortion incurred during aggregation and the release reaction.
Dihomo-γ-Linolenic Acid Metabolism in Man:Its Relevance to Platelet Function
Authors: Stone K. J. , Willis A. L. , Hart M. , Marples P.
Published: May 2019

Prostaglandin (PG) metabolites of dihomo-γ-linolenic acid (DELA) inhibit platelet aggregation (Kernoff et al, this meeting). In vivo, antithrombotic effects of DHLA might be limited by its rate of Δ5-desaturation to arachidonic acid (AA), which enhances platelet aggregation when administered to man. In the rat and mouse desaturation is particularly active but in the guinea-pig, cat and rabbit it appears insignificant*. We now report on the metaholism of single oral doses of DHLA in 3 human volunteers. Lipids were extracted from blood fractions and separated into different lipid classes before estimation of fatty acid composition by gas-liquid chromatographys P.G.’s, generated from platelets by maximal stimulation with thrombin, were isolated by argentation chromatography and estimated by bioassay. Maximal plasma DHLA/AA ratios (0.2-0.5) occurred at 3–4 hrs when the DHLA/AA ratio in triglycerides had increased 9–12 fold. Between 5-24 hrs DHLA accumulated in phosphatidyl choline. There were no consistent increases in the AA content of blood lipids and no increases in platelet production of its metabolite PGE2. Small increases in platelet PGE1 synthesis occurred, indicating that orally-administered DHLA reaches PG precursor pools in man. These results support our hypothesis that chronically-administered DHLA may be of therapeutic value as an antithrombotic agent.

*Willis, Stone, et al (1977). In: Prostaglandins in platelets and other blood cells, Ed. Silver, Smith and Kocsis, Spectrum Publications, Holliswood, N. Y.
Urinary Excretion of Factor VIII Related Antigen after Renal Transplantation
Authors: Ruggeri Z. M. , Coppola R. , Gordon Y. B. , Ardaillou N. , Ponticelli C. , Mannucci P. M.
Published: May 2019

Urinary excretion of factor VIII related antigen (VIIIR:AG) and fibrin(ogen) fragments (Fg) D and E was measured by radio-immunological techniques in unconcentrated urines obtained from 20 normal individuals, 20 patients (pts) followed serially after renal transplantation (tx) and 48 pts with constantly good renal function after tx. Pts serially followed showed a transitory excretion of VIIIR:AG, Fg D and E immediately after tx; subsequently, excretion was found only in connection with acute rejection episodes. Out of 20 episodes, VIII:AG was present in 16, Fg E in 13 and Fg D in 8, either accompanying or preceding of 1–2 days the increase of serum creatinine. In the group with good renal function the three proteins were occasionally present in urines but to a much lower extent than in patients with rejection episodes. Statistical analysis with the corrected X2 test showed a significant difference (P<0.001) between the two groups tor all the three measurements. The false positive rate (high excretion in patients with no rejection) was 6% for VIIIR:AG, 8% for Fg E and 10% for Fg D. Urinary excretion of VIIIR:AG proved to be more sensitive than Fg D and E as a parameter of kidney rejection, with a lower incidence of false positive results. This confirms that immunologically related endothelial damage and platelet aggregation precede intra-renal fibrin deposition and appearance of fibrin (ogen) degradation products in patients with acute rejection episodes.
Isolation of an Apparently Abnormal Von Willebrand Factor: Comparison with Plasma and Serum Von Willebrand Factor
Authors: Baugh R. F. , Burnand C. , Hougie C.
Published: May 2019

A patient with an extremely high level of von Willebrand factor (8X normal) failed to show any response to ristocetin with her platelet-rich plasma. The patient also had a long bleeding time and a large amount of a paraprotein. Using a washed platelet assay system and dilutions of her platelet-poor plasma, ristocetin-induced platelet aggregation activity could be recovered to 40% of normal. Purification of the von Willebrand factor from the patient’s plasma resulted in an increase in ristocetin-induced platelet aggregating activity associated with the von Willebrand factor. Electrophoretic comparison by SDS disc gel electrophoresis and crossed immunoelectrophoresis showed no differences between this patient’s von Willebrand factor and either purified normal plasma or serum von Willebrand factor. The patient’s plasma, following removal of the von Willebrand factor, inhibited ristocetin-induced platelet aggregation in normal platelet-rich plasma and in a washed platelet system using purified normal von Willebrand factor. These observations indicate the presence of an abnormal plasma protein which binds ristocetin and thereby inhibits platelet aggregation. Furthermore, the abnormal protein must interfere with the in vivo functions of the von Willebrand factor as witnessed by the increased bleeding time presented by the patient. This behavior is explained by a model system in which ristocetin mimics a structural component found in the lining of the vessel walls which is exposed by tissue injury. The abnormal plasma protein competes with the von Willebrand factor and interferes with von Willebrand dependent platelet adhesion.

Supported by an NIH Research Career Development Award and the American Heart Association.
Alteration of Coagulation in Intensively Transfused Hemophilic Patients
Authors: Hathaway Helen S. , Hamstra Roger D. , Jacobson Linda , Hathaway William E.
Published: May 2019

Bleeding may occasionally occur in adequately transfused hemophilic patients. To investigate this phenomenon, 11 patients with classical hemophilia had serial coagulation studies performed during intensive transfusion therapy with factor VIII concentrates given for surgical procedures. The studies included kaolin partial thromboplastin times (KPTT), fibrinogen, monomer, and fibrin split products (FSP) levels, and assays for factor VIII by the one-stage PTT method (PTT-VIII), thromboplastin generation time method (TGT-VIII), and immunologic method (VIII Ag). After an initial correcting dose, factor VIII concentrates were administered once to twice daily in a dose to keep the minimal level above 20 percent. Alterations of coagulation assays were most pronounced 7–10 days postoperatively. These included (1) KPTT values at least 20 seconds longer than expected for the percent factor VIII;(2) TGT-VIII levels consistently higher than PTT-VIII levels (mean difference was 20 percent);(3) VIII Ag values from 216–660 percent of normal. Fibrin monomer and FSP tests were frequently positive and fibrinogen levels ranged from 300–700 mgm percent. Four patients exhibited spontaneous wound bleeding on postoperative days 7, 7, 9, and 12 in spite of adequate factor VIII levels. These studies and the results of in vitro experiments with factor VIII concentrates suggest that altered proteins or degradation products of fibrinogen or factor VIII may produce spurious values for coagulation tests and may be associated with an increased bleeding tendency and/or abnormal wound healing.
Alterations of Plasmin Activity, Plasminogen Levels and Activity of Anti-Plasmins During Endotoxin Shock in Dogs
Authors: Aasen A. O. , Gallimore M. J. , Ohlsson K. , Amundsen E.
Published: May 2019

Endotoxin shock was induced in dogs by intravenous infusion of a lethal dose of E. coli endotoxin over a period of 3 hours. Typical changes of cardiovascular parameters were found and evidence of an intravascular clotting process was observed. Spontaneous plasmin activity and “immediate” and “time dependent” antiplasmin activities were determined by means of assays utilizing the chromogenic tripeptide derivative S-2251(Kabi Peptide Research Division, Mölndal, Sweden). Levels of plasminogen, α2-macrolobulin (α2-M) , and ai-antitrypsin(α1-AT) were determined immunochemically. During shock, gradually decreasing values of “immediate” antiplasmin and α2M were observed. During the late stages of shock “immediate” antiplasmin was found to be reduced by up to 89 per cent and α2M up to 50 per cent of pre endotoxin infusion values. A less marked lowering of “time dependent” antiplasmin and α1-AT also occurred during shock. These changes of plasma antiplasmins were accompanied by decreasing values of plasminogen and evidence of plasmin activity. These findings indicate that plasminogen is converted to plasmin during endotoxin shock and emphasize the role of antiplasmins in the pathophysiology of endotoxin shock.
Difference in Thrombolytic Effect between Higher and Lower Molecular Weight Forms of Urokinase
Authors: Suyama T. , Nishida M. , Iga Y. , Naito R.
Published: May 2019

Urokinase (UK) from human urine has been widely used for thrombolytic therapy in Japan. However, commercially available preparations are not identical but consist of mainly two forms of UK with higher and lower molecular weight (H-UK and L-UK) . An attempt was made in this report to compare thrombolytic activity of H-UK with that of L-UK on artificial thrombi produced from human blood by a modification of Chandler’s loop method, which was somehow comparable to the situation in vivo. Two active forms of UK were purified from crude preparation by gel filtration. The approximate molecular weight of the H-UK was 54,000 and of the L-UK 34,000. The potency of UK was determined by “two-stage lysis time method” and expressed by International unit(lU). Thrombolytic activity measured by Chandler’s method was calculated as % lysis of the control thrombus that was formed in the abscence of UK.

As a result, thrombus-dissolution time of H-UK was much shorter than that of L-UK. Furthermore, concentration of H-UK (IU/ml blood) necessary to induce 50% lysis was approximately one half lower than that of L-UK. The similiar results were obtained on artificial thrombi from the blood of dog, rat and rabbit. The data suggest that H-UK seems to be more effective on treatment of thromboembolicdisorders as compared to L-UK in terms of the same IU basis.
New Hypothesis for the Molecular Mechanism of Surface-Dependent Activation of Hageman Factor (Factor XII)
Authors: Griffin John H.
Published: May 2019

The surface-dependent mechanism of activation of highly purified human Hageman factor (HF) was studied using 3H-DFP uptake as a quantitative active site titrant. HF was treated in various ways and the reaction mixture was exposed to 3 mM 3H-DFP for 5 min at 37°. Following addition of SDS and removal of free DFP by dialysis, the reaction products were analyzed on SDS gels. In solution, the HF zymogen at 80,000 MW took up 0.015 mol DFP per mol HF. HF bound to kaolin, celite, or ellagic acid with or without high MW kininogen took up the same 0.015 mol DFP per mol HF. However, HF bound to celite or kaolin with high MW kininogen and kallikrein took up 0.9 mol DFP per mol HF into a 28,000 MW fragment of HF. In approximately half of these activated HF molecules, this 28,000 MW fragment was linked by disulfide bonds to a 52,000 MW fragment in a surface-bound 80,000 MW form of activated HF. In clotting assays, DFP did not inhibit kaolin-bound HF unless the surface-bound HF first had been proteolytically activated by kallikrein.

Kinetic studies of the cleavage of 125I-HF by kallikrein or by plasmin in the presence of high MW kininogen showed that kaolin-bound HF was cleaved more than 20 times faster than HF in solution.

These results suggest that binding to kaolin or celite or ellagic acid, classically known as “activating surfaces”, does not convert a detectable fraction (< 1%) of the bound HF molecules to active enzymes. Rather, surface-binding makes HF molecules much more susceptible to proteolytic activation in the presence of high MW kininogen, and the reciprocal proteolytic activations of HF and prekallikrein are thus greatly stimulated by “activating surfaces”.
Heterogeneity of the Factor VIII/Von Willebrand Factor Protein in Von Willebrand’s Disease
Authors: Gralnick H. R. , Sultan Y. , Coller B. S.
Published: May 2019

The recent advent of techniques to purify the factor VIII/von Willebrand factor (f.VIII/vWf) protein from plasma to quantitate the f.VIII/vWf protein and to measure the vWf (plasma ristocetin cofactor) have greatly added to our understanding of von Willebrand’s disease (vWd). The initial studies of antigen, procoagulant and vWf levels revealed a parallel reduction in all three activities in vWd suggesting a quantitative deficiency of the f. VIII/vWf protein and its biologic activities.

Recent studies, however, have suggested three major forms of vWd. The first group of patients have a quantitative defect with a parallel reduction of the f.VIII/vWf protein and of the antigen, vWf and procoagulant activities. The second group of patients appear to have a qualitative defect of the f.VIII/vWf protein. These individuals have normal levels of the antigen and procoagulant activity; however, the vWf activity is reduced or absent. The third group of patients have a combination of a qualitative and quantitative deficiency. These variants resemble both previous groups in that there are reduced levels of the antigen, procoagulant and vWf activities but usually greater reduction of the vWf activity.

Three major defects of the f.VIII/vWf protein have been recognized in vWd: 1) decreased plasma concentration of the f. VIII/vWf protein, 2) the apparent molecular weight of the protein is reduced and/or the largest molecular weight polymers are absent, and 3) there is a partial or total carbohydrate deficiency. For the f.VIII/vWf protein to express vWf activity, it must be of a minimal molecular size and have a specific carbohydrate content and/or sequence.
Immunological Arterial Injury in Atherogenesis
Authors: Minick C. R. , Williams L. R. , Alonso D. R.
Published: May 2019

Our experiments indicate that the synergy of immunological arterial injury resulting from either immune complex disease or graft rejection and hypercholesterolemia will lead to atherosclerosis. Repeated or protracted immunological arterial injury in synergy with more modest hypercholesterolemia, like that in Western man, leads to arterial lesions which closely resemble chronic human atherosclerosis. Sites of immunological arterial injury retain their increased propensity to accumulate lipid for weeks and months following the injury.

Indirect evidence from experiments of others suggests that increased endothelial permeability may be the primary event in some immunologically induced arterial injury. Results of our experiments indicate that endothelial damage and/or loss and platelet interaction with the arterial wall may be an early event in the pathogenesis of immunologically induced atherosclerosis. Moreover, our results indicate that at least in the instance of graft rejection, endothelial injury and platelet interaction may not only be an early event, but that continued injury and platelet interaction may contribute to progression of atherosclerosis.
Factor VIII (FVIII) Antibodies in Hemophilia
Authors: Shapiro S. S.
Published: May 2019

Antibodies to FVIII arise in some 10–15% of multitransfused patients with severe Hemophilia A. Specificity of the antibody for FVIII coagulant activity can be demonstrated by coagulation assays, normal bleeding times and FVIII antigen and Ristocetin cofactor levels, as well as by inability of the antibody to interfere with Ristocetin aggregation at levels completely neutralizing FVIII coagulant activity. A genetic factor in immune responsiveness has been suggested in the past, but never proven. Recent studies lend credence to this possibility. Two groups of antibody patients can be discerned. Some patients seem capable of synthesizing only low levels of antibody and such patients can be treated with FVIII. Others are strong responders and cannot be treated successfully with FVIII at the height of their immune response, but may benefit from FIX concentrates. In hemophiliacs, the majority of antibodies tested have been of the IgG class and predominantly or exclusively of a single light-chain type. Heavy-chain typing has shown an unusual representation of the IgG4 subclass. These observations suggest restricted heterogeneity of the immune response to FVIII. As shown by serial isoelectric focusing studies, the heterogeneity of antibody populations tends to vary markedly with time after antigenic stimulation, generally increasing with time after stimulation. This may explain some of the conflicting results in the literature on this subject. Immunosuppression has been of only limited usefulness in eliminating FVIII antibodies, and interpretation of “successful” cases is difficult because of the variable course of the antibodies.
The Role of Complement and Platelets in the Dissolution of Thrombin-Induced Clots
Authors: Taylor Fletcher B.
Published: May 2019

The dilute whole blood clot lysis assay has been used in diagnosis of patients with deep vein thrombophlebitis and pulmonary embolism. Because of its clinical value it has also been the subject of biochemical and physiologic studies of clot lysis of normal diluted blood. This assay reflects the behavior of platelets in that clot lysis as well as clot retraction are platelet dependent. Further, this contribution of platelets is temperature sensitive whereas the rate of fibrin clot formation is not. Thus, this assay offers a convenient model for functional and morphologic studies of temperature induced discontinuity of platelet-fibrin assembly and the interaction of platelet and fibrin in clot formation, retraction and lysis. In these studies the release of serotonin from platelets was correlated with clot formation, retraction, lysis, and clot morphology at 1) 37°, 4° and 4°−37° at 5, 15, 30 and 60 minute intervals. The results suggest that both platelets and fibrinogen are influenced by thrombin used in the assay and that under certain temperature conditions the platelets will release their contents out of phase with the assembly of the fibrin network. In cases where this discontinuity exists, the diluted clots will not retract or lyse normally.