bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein C Activity in Healthy Volunteers - Influence of Age, Sex, Smoking and Oral Contraceptives
Authors: Tait R C , Walker Isobel D , Islam S I A M , McCall Frances , Conkie J A , Wight M , Mitchell R , Davidson J F
Published: July 2018

Interpretation of protein C (PC) levels in a given individual has several limitations. A normal PC activity does not necessarily exclude a genetic deficiency nor can a reduced level confirm it. Measuring PC amidolytic activity in 9,648 healthy blood donors has allowed identification of demographic factors which cause variation in PC activity and further hinder interpretation. PC activity displays a log normal distribution and significant variation with age. This is most marked in young adult males when mean PC activity rises from 0.86 iu/ml (15–19 years) to 1.04 iu/ml (45–49 years; P <0.0001). Pre-menopausal females, who for most age ranges, have mean PC activity below their male contemporaries, show a less marked rise with age until the menopause when PC activity rises further. The use of hormonal contraceptive preparations is associated with an increase in mean PC activity of 0.05–0.08 iu/ml while smoking habit has no influence on PC activity.

In view of these findings we strongly recommend the use of age and sex restricted reference ranges when interpreting PC activity.
Thrombolytic and Haemorrhagic Effects of Bolus Doses of Tissue-Type Plasminogen Activator and a Hybrid Plasminogen Activator with Prolonged Plasma Half-Life (K2tu-PA: CGP 42935)
Authors: Agnelli Giancarlo , Pascucci Claudia , Nenci Giuseppe G , Mele Antonio , Bürgi Rolf , Heim Jutta
Published: July 2018

K2tu-PA is a hybrid plasminogen activator linking the kringle 2 domain of tissue-type plasminogen activator (t-PA) to the catalytic protease domain of single-chain urokinase-type plasminogen activator (scu-PA). K2tu-PA, as t-PA has high affinity for fibrin and is activated by fibrin but has a longer plasma half-life (over 30 min). The aim of this study was to compare the effects of bolus doses of recombinant t-PA (rt-PA) and K2tu-PA, on: 1) lysis of preformed thrombi (fibrinolysis), 2) accretion of new fibrin on pre-existing thrombi during fibrinolysis (thrombus growth), 3) thrombolysis as assessed by reduction of thrombus weight and 4) systemic plasma proteolysis and blood loss from a standard wound. A jugular vein thrombosis model and an ear bleeding model were adopted in rabbits. Saline produced 11 ± 2% fibrinolysis. rt-PA, 0.2 mg/kg, 0.4 mg and 0.8 mg/kg produced 35 ± 4%, 54 ± 4% and 78 ± 6% fibrinolysis, respectively. K2tu-PA, at the same doses, produced 39 ± 5%, 57 ± 6% and 83 ± 6% fibrinolysis, respectively. Thus, no differences in the fibrinolytic activity of rt-PA and K2tu-PA were observed. Injection of saline was followed by an accretion of 56.4 ± 5.9 μg of radioactive new fibrin on the thrombi. The injection of the three increasing doses of rt-PA was followed by an accretion of 54.9 ± 5.3 μg, 49.1 ± 6.1 μg and 47.2 ± 4.8 μg. The injection of three increasing doses of K2tu-PA was followed by an accretion of 38.1 ± 3.4 μg, 29.6 ± 2.5 μg and 17.1 ± 3.4 μg. At each of the three doses, K2tu-PA was more effective than rt-PA in reducing the accretion of new fibrin on the thrombi (p <0.01) and, as a consequence, in reducing thrombus weight (p <0.01). The two lower doses of rt-PA and K2tu-PA did not produce systemic proteolysis and bleeding. The highest dose of K2tu-PA produced a statistically significant more intense systemic proteolysis and bleeding than the highest dose of rt-PA.

This study demonstrates that bolus doses of K2tu-PA and rt-PA produce a similar degree of fibrinolysis. Due to its longer half-life K2tu-PA is more efficient than rt-PA in inhibiting accretion of new fibrin on the thrombi during thrombolysis so that the thrombus size is more efficiently reduced. As a consequence the concomitant use of heparin might not be necessary. The potential increased risk of bleeding with bolus of high doses of K2tu-PA has to be seen in view of the advantage of avoiding the concomitant use of heparin.
The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis
Authors: Robbie Linda A , Booth Nuala A , Croll Alison M , Bennett Bruce
Published: July 2018

The relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.
A Slow Clearing, Fibrin-Specific, PAI-1 Resistant Variant of t-PA (T103N, KHRR 296-299 AAAA)
Authors: Paoni Nicholas F , Keyt Bruce A , Refino Canio J , Chow Alice M , Nguyen Hung V , Berleau Lea T , Badillo Julie , Peña Luis C , Brady Kevin , Wurm Florian M , Ogez John , Bennett William F
Published: July 2018

Site directed mutagenesis was used to construct a t-PA variant that contains an additional glycosylation site in the first kringle domain (T103N) combined with a tetra-alanine substitution in the protease domain (KHRR 296-299 AAAA). This combination variant has a plasma clearance rate that is 4.5-fold slower in rats and 5.4-fold slower in rabbits than t-PA. It is also less than one tenth as active as t-PA towards plasminogen in the presence of fibrinogen, and has approximately twice the normal activity in the presence of fibrin. It shows substantial resistance to the fast acting inhibitor, plasminogen activator inhibitor-1 (PAI-1), requiring a 10-fold greater molar excess of PAI-1 to reduce its activity by 50%, compared to t-PA. This is the result of a reduction of nearly 100-fold in the second order rate constant for PAI-1 inactivation. These results show that it is possible to combine mutations in different domains of t-PA to construct a variant which is simultaneously slower clearing, less reactive towards plasminogen in the absence of a fibrin clot, and resistant to inactivation by PAI-1.
Functional Properties of p-Anisoylated Plasmin-Staphylokinase Complex
Authors: Lijnen H R , Van Hoef B , Smith R A G , Collen D
Published: July 2018

The kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p’-anisate-HCI was 52 M-1 s-1 and its deacylation rate constant 1.2 × 10-4 s-1 (t½ of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min.

The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.
Effect of Diltiazem and Low-Dose Aspirin on Platelet Aggregation and ATP Release Induced by Paired Agonists
Authors: Zucker Marjorie L , Budd Susan E , Dollar Lawrence E , Chernoff Steven B , Altman Raul
Published: July 2018

The authors studied the effects of diltiazem, administered alone and together with low-dose aspirin, on the platelet response to paired agonists. After a baseline period, 25 healthy volunteers were given oral diltiazem for 1 week (120, 240, or 360 mg/day), and then crossed over randomly between 1 week on diltiazem plus aspirin (81 mg/day), and 1 week on aspirin (81 mg/day) alone. Platelet function was tested on 2 consecutive days in each period. Synergistic platelet aggregation and ATP release were obtained at baseline using a subthreshold concentration of arachidonic acid combined with platelet activating factor, ADP, or epinephrine. Diltiazem resulted in a significant decrease from baseline in platelet aggregation and ATP release using the arachidonic acid-epinephrine combination (35% and 40% decrease, respectively, p <0.01) and a significant decrease in aggregation using the arachidonic acid-ADP combination (22% decrease, p <0.01). The effects were neither dose-related, nor accompanied by any significant change in serum thromboxane B2 levels or bleeding times. There was no significant difference between the effects of aspirin alone and aspirin plus diltiazem on the synergistic platelet aggregation and ATP release induced by the paired agonists, or on thromboxane B2 levels or bleeding times. Diltiazem administered in vivo partially inhibits the synergistic platelet aggregation and ATP release induced by paired agonists; however, in contrast to a previous in vitro study it does not potentiate the platelet-inhibitory effect of aspirin.
Platelet Activating Properties of Murine Monoclonal Antibodies to β2-Glycoprotein I
Authors: Arvieux J , Roussel B , Pouzol P , Colomb M G
Published: July 2018

Previously developed murine monoclonal antibodies (MAbs) to human β2-glycoprotein I (β2GPI), a plasma protein required for the binding of anti-phospholipid antibodies, were studied for anti-platelet reactivity and influence on platelet function. The six MAbs (IgG1 isotype) tested interacted with both intact and fixed platelets in a β2GPI-dependent manner. Carbamylated β2GPI was still recognized by MAbs but was unable to mediate platelet- antibody binding. MAbs induced aggregation and secretion responses of platelets in platelet-rich plasma (PRP) and whole blood, provided subthreshold concentrations of weak agonists (i.e. ADP or adrenaline) were added. When aggregation in PRP was evaluated by a counting technique instead of turbidometrically, the sole addition of MAbs led to a rapid fall in single platelets. Triggering gel-filtered platelets with MAbs together with β2GPI, but not its carbamylated form, led to platelet activation after a lag time, as monitored by aggregometry, measurements of ATP and β-thromboglobulin secretion and calcium mobilization. F(ab')2 fragments of one of the MAbs failed to activate platelets but inhibited the responses to the whole antibody. This process thus depends on MAbs binding to platelets through both Fab and Fc domains, as confirmed by the suppression of platelet responses upon pretreatment with the anti-FcγRII MAb IV.3. Aggregation and secretion induced by MAbs plus β2GPI did not require exogenous fibrinogen and were variably inhibited in the presence of acetyl salicylic acid, apyrase or Ca2+, depending on the concentrations used for the two proteins. We propose that platelet FcγRII crosslinking that follows a platelet-antibody interaction via the platelet binding protein, β2GPI, may be a new mechanism by which anti-β2 GPI antibodies activate platelets and/or synergize with weak agonists.
A Quantitative Protein S Deficiency Associated with a Novel Nonsense Mutation and Markedly Reduced Levels of Mutated mRNA
Authors: Yamazaki Tomio , Hamaguchi Motohiro , Katsumi Akira , Kagami Kazuo , Kojima Tetsuhito , Takamatsu Junki , Saito Hidehiko
Published: July 2018

A 50-year-old Japanese man who had experienced recurrent episodes of venous thrombosis was found to have a hereditary protein S deficiency. The amount of total protein S antigen in plasma was reduced by approximately 50% in the patient and his two sons. DNA sequence analysis revealed a novel nonsense mutation, TAG for Gin 522 (CAG), in exon 14 of the protein S gene. Family studies performed by mutagenic PCR followed by restriction enzyme digestion showed that the proband and his two sons were heterozygous for this mutation. An mRNA-based analysis indicated that transcripts of the mutated allele were markedly reduced in the platelets of the affected individuals. Immunoblot analysis failed to detect the truncated mutant of protein S in the plasma or platelets of affected members. Our results demonstrated that this novel nonsense mutation was responsible for the quantitative deficiency of protein S.
Pre-operative Plasma Levels of Thrombin-Antithrombin III Complexes Correlate with the Development of Venous Thrombosis after Major Hip or Knee Surgery
Authors: Ginsberg Jeffrey S , Brill-Edwards Patrick , Panju Akbar , Patel Ameen , McGinnis Joanne , Smith Frank , Dale Ian , Johnston Marilyn , Ofosu Fred
Published: July 2018

Study objective. To determine whether levels of thrombin-antithrombin III (TAT) in plasma, taken two weeks pre-operatively, predict the development of deep vein thrombosis (DVT) in patients undergoing major hip or knee surgery.

Design. Prospective cohort.

Setting. Tertiary-care referral centre, university-affiliated hospital.

Patients. Ninety eight consecutive patients undergoing elective hip or knee surgery.

Intervention. All eligible consenting patients were seen in a preoperative clinic two weeks prior to surgery and had blood taken for measurement of plasma TAT level. After surgery, they received a combination of unfractionated heparin 5000 Units 12-hourly subcutaneously, and antiembolism stockings (TEDS), as prophylaxis against DVT. Contrast venography was performed prior to discharge, and according to the results, patients were classified as having proximal (popliteal and/or more proximal) DVT (n = 12), calf DVT (n = 7) or no DVT (n = 79).

Measurements and Results. The mean TAT level was significantly higher in patients who developed DVT (5.7 μg/l) than in those who did not (4.1 μg/l), p = 0.035. Using cut-points of 3.5 and 5.5 μg/l for the TAT level, patients could be categorized as high, intermediate, and low risk for the development of DVT. The proportion of patients with TAT levels of ≥3.5μg/l who developed calf or proximal DVT was significantly higher than the proportion of patients with TAT levels of <3.5 μg/l who developed calf or proximal DVT (p = 0.02). The proportion of patients with TAT levels >5.5 μg/l who developed proximal DVT was significantly higher than the proportion of patients with TAT levels of ≤5.5 μg/l who developed proximal DVT (p = 0.03).

Conclusions. This study demonstrates that pre-operative TAT levels correlate with the risk of developing DVT after major orthopedic surgery. Further studies are needed to determine the reason(s) for this observation and whether rational recommendations about prophylaxis and screening for DVT can be made based on the results of a pre-operative TAT level.
Endothelial Haemostatic Factors May Be Associated with Mortality in Patients on Long-term Anticoagulant Treatment
Authors: Brännström Mats , Jansson Jan-Håkan , Boman Kurt , Nilsson Torbjörn K
Published: July 2018

The aim of the present study was to test if long-term mortality could be predicted by endothelial derived haemostatic variables in a population with high morbidity due to thromboembolic disease. Plasma samples were drawn from 212 out-patients treated with oral anticoagulants, at the beginning of the study, and analyzed for mass concentration of tissue plasminogen activator (tPA) and its inhibitor (PAI-1), and von Willebrand factor. In the course of 3.8-year follow-up 45 patients died, including 38 vascular deaths. We found that all-cause mortality was significantly associated with increased levels of vWF and tPA. For vascular mortality there was a significant association with all three haemostatic variables (tPA, PAI-1, vWF). For vWF there was a 3-fold increase in total and vascular mortality in the highest quartile compared to the lowest quartile. There were 27 vascular deaths in the group of patients with a tPA-value above the median compared to 11 in those with a tPA below the median. In multivariate Cox regression analysis (including: age, sex, smoking habits, body mass index, diabetes mellitus, hypertension, tPA, PAI-1, and vWF), vWF and smoking were independently significantly associated with all-cause mortality, and tPA and age with vascular mortality. Endothelial derived haemostatic variables are predictors of total and vascular mortality in patients treated with oral anticoagulants.
Pharmacokinetics and Tolerability of Factor XIII Concentrates Prepared from Human Placenta or Plasma: a Crossover Randomised Study
Authors: Brackmann H H , Egbring R , Ferster A , Fondu P , Girardel J M , Kreuz W , Masure R , Miloszewski K , Stibbe J , Zimmermann R , Krzensk U , Hoos A
Published: July 2018

The pharmacokinetics and tolerability of factor XIII (FXIII) from plasma were compared with those of FXIII from placenta in a randomised, double-blind, crossover study involving 13 patients with congenital FXIII deficiency. Both FXIII activity and FXIII antigen were monitored. No difference was seen in the mean half-lives of the two preparations (9.3 days and 9.1 days for plasma and placenta FXIII activity, respectively). Response was similar for both preparations, but was slightly greater for FXIII from plasma.

Similar results were found for recovery (65% vs 60%). The area under the data completed by extrapolation was significantly higher for FXIII from plasma. No differences between preparations in terms of efficacy or tolerability were observed. It can be concluded that treatment with FXIII concentrate from plasma is as efficient as with FXIII concentrate from placenta in terms of recovery and half-life. Both preparations were equivalent in terms of safety during the observation period. With the administration of monthly injections of approximately 30 U/kg serious bleeding events were prevented and no other serious adverse events occurred.
Pharmacokinetics of Heparin in Healthy and Obese Subjects and in Combination with Dihydroergotamine
Authors: Beermann Björn , Lahnborg Gordon
Published: July 2018

Heparin (5000 IE s.c., 5000 IE i v, 200 IE/kg i v) was given to healthy volunteers without and with 0.5 mg dihydroergotamine (DHE) i v to test whether DHE influences the kinetics of heparin. The anticoagulant (200 IE/kg) was also administered to obese subjects. Heparin was assayed using a chromogenic substrate for activated factor X.

The plasma levels of heparin did not differ whether given concomitantly with DHE or not. Halflife of heparin was also uninfluenced of DHE demonstrating that the beneficial effect of DHE on postoperative thromboembolism is not caused by interaction with the pharmacokinetics of heparin.

The plasma levels of heparin were significantly higher in the obese subjects than in the normal ones. The halflife of the drug averaged 2.13 h in the obese subjects and 1.36 h in the normal subjects (p<0.02). It was concluded that the heparin dosage should be calculated in terms of estimated ideal weight.
The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography
Authors: Kavanagh M L , Wood C N , Davidson J F
Published: July 2018

Nine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.
Influence of ω-Aminoacids on the Activation of the First Component of Complement at Low Ionic Strength
Authors: Takada Yumiko , Takada Akikazu
Published: July 2018

Cl, the first component of human complement, was activated to Cls by diluting human plasma in a solution of low ionic strength. The addition of lysine and its related aminoacids to plasma resulted in inhibition of Cl activation, α, ω-diaminoacids such as lysine and ornithine were most effective and ω-aminoacids such as 6 aminohexanoic acid were far less effective. Tranexamic acid which is most potent in inhibition of fibrinolysis among those aminoacids was least effective in the inhibition of Cl activation, ω-aminoacids or their analogues hardly inhibited Cls which had already been activated.
Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen
Authors: Sueishi Katsuo , Nanno Shigeru , Tanaka Kenzo
Published: July 2018

Fibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observation of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction.

These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.
Contribution of Plasma Proteinase Inhibitors to the Regulation of Activated Protein C in Plasma
Authors: Marlar Richard A , Kressin David C , Madden Renee M
Published: July 2018

Activated protein C (APC), a serine protease, is regulated in plasma by protease inhibitors. This study was undertaken to determine the role of the major plasma inhibitors in regulating APC in plasma. Kinetic analysis and specific immunoassays for APC-inhibitor complexes were used to determine the inhibitors that form complexes with APC. Of the eight plasma inhibitors investigated, four interact with APC: protein C inhibitor (PCI), α1-proteinase inhibitor (PI), α2-antiplasmin (AP) and C1 esterase inhibitor (C1 Inh). The second order rate constants are: 1.3 × 104 M−1 s−1 (PCI); 15 M−1 s−1 (PI); 410 M−1 s−1 (AP); and <6 M−1 s−1 (C1 Inh), with a relative effectiveness of each inhibitor to inactivate APC in plasma: 49:36:15:<1, respectively. PCI, PI and AP are the major inhibitors of APC in plasma. Low concentrations of APC will be inhibited by PCI with PI and AP playing a secondary role. However, as increasing APC is generated, PI and AP begin to play more important roles as the PCI is consumed.
Relations between Activation and Inhibition of Fibrinolysis in the Walls of Human Arteries and Veins
Authors: Hegt Vincent Noordhoek
Published: July 2018

From systematic studies of both the activation and the inhibition of fibrinolysis in human blood vessels, data which provided insight into the fibrinolysis antagonism along and across the human vessel wall were obtained.

The capacity for fibrinolysis initiated by plasminogen activator in sections of human arteries and veins as demonstrated by the fibrin sfide technique differed greatly along and across the vessel walls. Inhibition of plasmin fibrinolysis in these blood vessels as detected by the fibrin slide sandwich technique was present at sites of low fibrinolytic activity, while inhibition was absent in areas showing strong fibrinolysis. Fibrinolysis was related to endothelial cells, while inhibition of fibrinolysis was brought about by smooth muscle cells. The results indicate that differences in endothelial fibrinolysis may be the result of differences in inhibition of fibrinolysis caused by variations in the number of smooth muscle cells present locally. A systemic decrease in endothelial fibrinolysis observed in endotoxin shock, hyaline membrane disease and a Waterhouse-Friderichsen syndrome appeared to be associated with a systemic increase in inhibition of fibrinolysis not related to smooth muscle cells.
The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study
Authors: Tinlin Shawn , Webster Sandra , Giles Alan R
Published: July 2018

The development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.
Multi-Center Study of Thromboplastin Calibration Precision – Influence of Reagent Species, Composition, and International Sensitivity Index (ISI)
Authors: Besselaar A M H P van den , Bertina R M
Published: July 2018

Four thromboplastin reagents were tested by 18 laboratories in Europe, North-America, and Australasia, according to a detailed protocol. One thromboplastin was the International Reference Preparation for ox brain thromboplastin combined with adsorbed bovine plasma (coded OBT/79), and the second was a certified reference material for rabbit brain thromboplastin, plain (coded CRM 149R). The other two thromboplastin reagents were another rabbit plain brain thromboplastin (RP) with a lower ISI than CRM 149R and a rabbit brain thromboplastin combined with adsorbed bovine plasma (RC). Calibration of the latter two reagents was performed according to methods recommended by the World Health Organization (W. H. O.).

The purpose of this study was to answer the following questions: 1) Is the calibration of the RC reagent more precise against the bovine/combined (OBT/79) than against the rabbit/plain reagent (CRM 149R)? 2) Is the precision of calibration influenced by the magnitude of the International Sensitivity Index (ISI)?

The lowest inter-laboratory variation of ISI was observed in the calibration of the rabbit/plain reagent (RP) against the other rabbit/plain reagent (CRM 149R) (CV 1.6%). The highest interlaboratory variation was obtained in the calibration of rabbit/plain (RP) against bovine/combined (OBT/79) (CV 5.1%). In the calibration of the rabbit/combined (RC) reagent, there was no difference in precision between OBT/79 (CV 4.3%) and CRM 149R (CV 4.2%). Furthermore, there was no significant difference in the precision of the ISI of RC obtained with CRM 149R (ISI = 1.343) and the rabbit/plain (RP) reagent with ISI = 1.14. In conclusion, the calibration of RC could be performed with similar precision with either OBT/79 or CRM 149R, or RP.

The mean ISI values calculated with OBT/79 and CRM 149R were practically identical, indicating that there is no bias in the ISI of these reference preparations and that these reference preparations have been stable since their original calibration studies in 1979 and 1987, respectively.

International Normalized Ratio (INR) equivalents were calculated for a lyophilized control plasma derived from patients treated with oral anticoagulants. There were small but significant differences in the mean INR equivalents between the bovine and rabbit thromboplastins. There were no differences in the interlaboratory variation of the INR equivalents, when the four thromboplastins were compared.
Effect of Heparin on Thrombin Inhibition in the Microcirculation
Authors: Nydahl S , Frebelius S , Swedenborg J
Published: July 2018

Disappearance of thrombin enzymatic activity was measured during recirculation through the microvasculature in a rat Langendorff heart preparation. This resulted in a 50% loss of thrombin from the recirculating solution. No increased loss of thrombin could be demonstrated if a mixture of antithrombin III and thrombin was recirculated, compared to thrombin alone. If, however, a heparin/thrombin mixture was recirculated, a 90% loss of thrombin could be demonstrated. Pretreating the microvasculature with large amounts of heparin resulted in recovery of antithrombin III in the recirculated heparin solution. At a subsequent recirculation with a heparin/thrombin mixture the loss of thrombin was decreased to the control level, as seen when recirculation with thrombin alone was performed. It is concluded that disappearance of thrombin enzymatic activity from a solution when recirculated through the microcirculation can be considerably increased if recirculated together with heparin, which probably reacts with endogenous antithrombin III on the vessel wall. The disappearance of thrombin in the absence of heparin was, however, unaffected by antithrombin III. The latter finding is compatible with the hypothesis that, in the microcirculation, antithrombin III/glycosaminoglycans play only a minor role for inhibition of thrombin coagulant activity and that thrombin binds mainly to thrombomodulin.
Spontaneous Atrial Thrombosis in Aged Syrian Hamsters II. Hemostasis
Authors: Dodds W. Jean , Raymond Sharon L. , Moynihan Ann C. , McMartin Donald N.
Published: July 2018

Coagulation and fibrinolysis were studied in a colony of aged Syrian hamsters with spontaneous atrial thrombosis, and the results are consistent with concomitant consumption coagulopathy. In comparison to age- and sex-matched hamsters from the same colony, those with atrial thrombi had significantly prolonged prothrombin and partial thromboplastin times, reduced levels of factors II, VII, VIII and X activities and plasminogen; and concentrations of fibrinogen-fibrin split products in excess of 80 μg/ml. Hematocrits of the thrombosed animals were significantly decreased, total plasma proteins were increased, leukocyte counts were within normal limits, and platelet counts were about half those of the controls. Thrombosed hamsters had significantly reduced plasma albumin content, increased γ1-, β-, and γ-globulins, and reduced A/G ratios.

Aged sick hamsters without demonstrable thrombi also had reduced coagulation and fibrinolytic activities and platelet counts, but their fibrinogen levels were markedly elevated, and fibrinogen-fibrin split products were either absent or present in trace amounts. This suggests an earlier and/or less acute form of the thrombotic process.
Antithrombin III and Heparin Inactivation in Thrombin Involving Reactions
Authors: Marciniak Ewa
Published: July 2018

The inhibitory capacity of antithrombin III (AT III) was measured by a quantitative method independent of the velocity of inhibition. When AT III was in excess of thrombin in plasma or in purified system the capacity of inhibitor decreased quantitatively in proportion to the amount of thrombin neutralized. Heparin present in reaction together with thrombin invariably induced a more extensive utilization of inhibitor than thrombin alone. The extent of this additional loss of inhibitory capacity was to a limited degree related to the concentration of heparin. Heparin itself was neutralized in thrombin-AT III reaction losing its anticoagulant property in proportion to the amount of thrombin bound by inhibitor. This quantitative neutralization of heparin occurred not only when the anticoagulant participated in thrombin-AT III binding but also when heparin was added to a medium containing a preformed thrombin-AT III complex. These results suggest that acceleration of binding and increased utilization of binding capacity are the two regular effects of heparin on thrombin-involving reactions of AT III. Both of these effects may be abolished by quantitative binding of heparin to thrombin-AT III complex.
The Different Forms of Antithrombin III in Serum
Authors: Pepper D. S , Banhegyi D , Cash J. D
Published: July 2018

Antithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.

Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.
The Aspirin Tolerance Test in von Willebrand’s Disease
Authors: Barbui Tiziano , Rodeghiero Francesco , Dini Enrico
Published: July 2018

Opinions about the clinical value of aspirin tolerance test proposed by Quick in von Willebrand’s disease are conflicting.

The results of the present study seem to support the view that a platelet defect, induced by aspirin, causes a significant lengthening of bleeding time only in the cases with severe von Willebrand factor deficiency.
Fibrin and Fibrinogen Proteolysis Products: Comparison between Gel Filtration and SDS Polyacrylamide Electrophoresis Analysis
Authors: Alkjaersig Norma , Davies Andrew , Fletcher Anthony
Published: July 2018

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400,000 to 800,000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and nonstabilized fibrin in that fragment D was present in the “double D” cross-linked form.
Alteration in Second Phase Platelet Aggregation Associated with an Emotionally Stressful Activity
Authors: Arkel Yale S. , Haft Jacob I. , Kreutner William , Sherwood Joseph , Williams Rae
Published: July 2018

23 healthy house staff officers were studied for platelet aggregation changes immediately prior to, immediately after and at a 7 to 11 day interval after their presenting a case before the Medical Mortality Conference. In 12 of the 23 with epinephrine (2.5 × 10−6M) and 11 of the 23 with ADP (2.0 × 10−6M) an absence of second phase aggregation was noted. A total of 19 of the 23 with epinephrine (2.5 × 10−6M) either had absent second phase or had a decreased slope of second phase aggregation. In all subjects except two a return to normal pattern was noted in the recovery samples 7 to 11 days later.

In 5 subjects who had absent second phase aggregation with epinephrine (2.5 × 10-6M) immediately after presentation, one had a return toward normal at 24 hours while the others were resistant to higher concentrations of epinephrine (2.5 × 10−5M and 2.5 × 10−4M). One subject in the immediate post presentation period had a slight improvement in second phase aggregation with 2.5 × 10−4M epinephrine.

Platelet counts increased in 5 of 6 subjects in the immediate post presentation period and did not necessarily correlate with the absence of second phase aggregation.

ADP/ATP platelet content increased in the post presentation samples.

We can conclude that during and immediately after an activity associated with stress, platelet changes can occur characterized by a decreased second phase aggregation with epinephrine or ADP. These changes last for at least 24 hours and are resistant to higher concentrations of epinephrine.
Fibrinogen, Fibrinogen Degradation Products and Erythrocyte Flexibility
Authors: Rampling Michael W. , Sirs John A.
Published: July 2018

An investigation has been made of the effect of fibrinogen concentration and fibrinogen degradation products on the flexibility of erythrocytes. The measurements were made using the blood of 2 donors, one representative of the normal population, the other representative of the 1. 5% of the normal population whose flexibility is more than three standard deviations from the total population mean. A linear relationship between erythrocyte flexibility and fibrinogen concentration was found in both cases. However, while the normal control had only a slow packing rate at zero fibrinogen concentration, the fast-packing cells retained a high degree of flexibility. Digestion of fibrinogen for sufficient time to form the larger degradation fragments X and Y indicated that they have similar effects to the native fibrinogen. The smaller fragments D and E, do not affect the flexibility nor do they inhibit or modify the effect of fibrinogen.
Heparin Neutralizing Activity (HNA) and Antithrombin III in Coronary Artery Disease
Authors: Cella G , Russo R
Published: July 2018

Plasma from 14 patients with severe and diffuse coronary atherosclerosis has been compared with that obtained from a normal control group. While a decreased he parin-thrombin clotting time was demonstrated in the patient group, suggesting an increased level of circulating platelet factor 4, there was no significant alteration in plasma antithrombin III level. These results do not support a recent suggestion of a mild chronic intravascular coagulation in atherosclerosis.
Studies on the Neutralization of Rabbit Antibodies to Human Factor VIII
Authors: Kernoff P. B. A , Kaelin A. C
Published: July 2018

The capacity of normal and haemophilic cryoprecipitates to neutralize the anticoagulant effect of rabbit antibodies to human factor VIII (anti-VIII) was assessed using a quantitative estimation of antibody. About 4 times as much anti-VIII could be neutralized by normal factor VIII as was required to neutralize clotting activity. This suggests that there are probably several antigenic sites intimately associated with factor VIII clotting activity, quite apart from any antigenic sites which may be detected using antibodies directed against other components of the factor VIII complex. The neutralizing capacity of factor VIII was only slightly greater for the rabbit antibodies employed in this study than has been previously reported for antibodies of human origin, thus indicating only minor differences in specificities. Additional evidence in support of this concept was the finding that cryoprecipitates prepared from haemophilic plasmas previously recognized as either lacking or possessing the capacity to neutralize antibodies of human origin neutralized least or most quantities of rabbit antibodies, respectively.
A) Coagulation Proteins and Inhibitors: Cold-Insoluble Globulin (Clg), a Circulating Cell Surface Protein
Authors: Mosesson Michael W.
Published: July 2018

The history of the discovery and characterization of the cold-insoluble globulin of plasma (Clg) is reviewed. Subsequent growth of knowledge relating to its structure and function in plasma, in tissues, and on cell surfaces is examined.
Serine Protease Specificity for Peptide Chromogenic Substrates
Authors: Mattler L. E , Bang N. U
Published: July 2018

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HC1), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HC1), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HC1) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HC1) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa.

S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HC1) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
Plasma Antithrombin III and Factor VIII Antigen in Relation to Angiographic Findings, Angina and Blood Groups in Middle-Aged Men
Authors: Stormorken Helge , Erikssen Jan
Published: July 2018

In a prospective study on latent coronary heart disease (CHD) in 2,014 presumably healthy men aged 40–59 years, 115 had strong clinical evidence of CHD never diagnosed nor suspected prior to the survey. Coronary angiography was performed in 105 of these. In 80 of them – whereof 57 were angiopositive and 23 angionegative – AT III and factor VIII Ag were assayed as well as in 35 normals from the same material. The results showed that:

1. AT III was significantly lower in the angiopositive than in the normal controls.

2. Subdivision of the angiopositive into those with and those without angina pectoris revealed significantly lower level of AT III in the former.

3. Within blood group A only did the angiopositive subjects have significantly lower AT III than the remaining subjects in the group, and this was entirely due to the low values in those with angina pectoris.

4. Blood group 0 individuals had significantly lower factor VIII Ag level than all other blood groups.

The significance of these findings for the possible relation to development of atheromatosis is briefly discussed.
Measurement of Nucleotide Pools in Platelets Using High Pressure Liquid Chromatography
Authors: D’Souza Leo , Glueck Helen I.
Published: July 2018

We have devised an improved high pressure liquid chromatographic technique whereby serotonin, nucleosides, cyclic nucleotides, namely cAMP and cGMP, and 5’ mono-, 5’ di-, and 5’ tri-nucleotides can be analyzed. The cyclic nucleotides have been measured in pico molar quantities. All nucleotides can be quantitated in a single step separation in 75 min using a 0.0015 M phosphoric acids vs. 1 M pH 4.8 ammonium phosphate gradient. 5/10 ml of platelet-rich plasma furnishes an adequate sample for complete analysis. Nucleotide levels in platelets from 16 normal donors expressed in 1011 platelets are as follows: cAMP, 6.32 (4.15) nanomoles and AMP, 0.32(0.14); ADP, 2.48 (0.67); ATP 3.78 (0.68); GDP 0.38 (0.07) and GTP, 0.45 (0.07) micromoles. ADP and ATP values are lower than those previously published. However, the total nucleotide level approaches published values.

Upon aggregation with thrombin, approximately 50% of ADP and 40 % ATP is released. Release is complete by 2 min. Thrombin is the most potent releasing agent with collagen and ADP occupying an intermediate role and epinephrine being the least effective. Upon aggregation cyclic AMP levels diminish along with other nucleotides. Patients with asthma showed depression of ADP, ATP, GDP and GTP levels.