bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of Platelet Functions by Monobromobimane, a Fluorescent Thiol Group Label
Authors: Zucker Marjorie B , Mauss Evelyn A
Published: July 2018

Monobromobimane (mBBr, bimane), a compound that penetrates cells and forms a fluorescent adduct with thiol groups, was used to assess the significance of thiols in platelet function. Exposure of washed platelets for 1 min to 100 μM mBBr abolished ADP-induced aggregation; shape change was not inhibited by 500 μM mBBr. The nonpenetrating compound monobromotrimethylammoniobimane was ineffective. Established ADP-induced aggregation was reversed by bimane, and fibrinogen binding to ADP-stimulated platelets was inhibited, an effect mainly due to decreased number of binding sites. Aggregation stimulated by A23187 and arachidonate was less effectively inhibited whereas epinephrine- and collagen-induced aggregation were abolished by 50 μM mBBr. Similar effects on aggregation and secretion were observed in platelet-rich plasma except that higher mBBr concentrations were usually necessary. Aggregation and 14C-serotonin secretion stimulated by 0.1 U/ml thrombin were partially inhibited by pretreatment with bimane. With lower thrombin concentrations, they were often enhanced, as was 3H-arachidonate release. Bimane inhibited epinephrine-induced arachidonate release in gel-filtered platelets, possibly because it abolished the primary aggregation necessary for this release. mBBr did not elevate cyclic AMP but enhanced the increase induced by PGEi and prevented the subsequent decrease typically caused by ADP. Examination of SDS polyacrylamide gels with ultraviolet light showed that mBBr reacted with many platelet proteins but not with GP lib or Ilia. This observation, and the fact that bimane did not inhibit the fibrinogen-induced aggregation of DTT- or chymotrypsin-treated platelets suggest that it reacts with thiol group(s) that are involved in “exposing” the fibrinogen receptor.
Glycoproteins IIb and IIIa and Platelet Thrombospondin in a Liposome Model of Platelet Aggregation
Authors: Rybak M E
Published: July 2018

Platelet membrane glycoproteins IIb and IIIa and platelet thrombospondin were incorporated onto phosphatidylcholine liposomes, by freeze thawing and sonication. Protein orientation on the liposomes was confirmed by susceptibility to neuraminidase cleavage and binding to lentil lectin-Sepharose (GPIIb-IIIa liposomes) and to heparin-Sepharose (thrombospondin liposomes). Glycoproteins Ilb-IIIa bound 125I-fibrinogen with Kd of 7.5 × 10™7M. Binding was reversible and calcium-dependent. Ilb-IIIa liposomes underwent fibrinogen-dependent aggregation in the presence of 10 mM CaCl2. Maximal aggregate formation was observed with a combination of IIb-IIIa liposomes and thrombospondin liposomes. This aggregation was partially inhibited by preincubation with monoclonal antibodies to the IIb-IIIa complex. Addition of EDTA caused complete reversal of aggregates. Thrombospondin liposomes also underwent fibrinogen and calcium dependent aggregation, however, this aggregation was less than that observed with the GPIIb-IIIa liposomes. Maximal aggregate formation was observed with a mixture of IIb-IIIa liposomes and thrombospondin liposomes. These studies demonstrate that GPIIb-IIIa and thrombospondin can be incorporated into phospholipid vesicles with preservation of function. Direct evidence is provided to demonstrate that glycoprotein lib and Ilia and fibrinogen are sufficient for platelet aggregation and to demonstrate that thrombospondin may also contribute to platelet aggregation.
Immunological Evidence for Prothrombin in Human Platelets
Authors: Alexander R J
Published: July 2018

An attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma (3). Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it i) reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, ii) adsorbed a protein from the supernatant solution of activated platelets, and iii) inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that 1) plasma does not contain appreciable amounts of GPV, and 2) platelets contain prothrombin or an immunochemically similar protein.
The Anticoagulant Effect of Heparinoid Org 10172 During Haemodialysis: An Objective Assessment
Authors: Ireland Helen , Lane D A , Flynn Angela , Anastassiades E , Curtis J R
Published: July 2018

The heparinoid of natural origin Org 10172 has anti-factor Xa activity but minimal anti-thrombin activity, and little effect upon broad spectrum assays such as the KCCT in vitro. Its anticoagulant effects have been compared to those of commercial heparin in 7 patients undergoing haemodialysis for chronic renal failure. Commercial heparin was administered in a dose (5,000 iu bolus + 1,500 iu/hour continuous iv infusion) previously shown to inhibit fibrin formation during haemodialysis. This produced mean anti-factor Xa levels in plasma between 0.7-1.0 iu/ml and largely suppressed fibrin formation for 5 h dialysis measured as mean FPA levels in plasma. Administration of Org 10172 as a bolus of 1,350 anti-factor Xa u or 2,000-2,400 anti-factor Xa u produced plasma anti-factor Xa levels of less than 0.5 u/ml and allowed fibrin clot and FPA generation during dialysis. Org 10172 administered as a bolus dose of 4,000-4,800 anti-factor Xa u produced mean anti-factor Xa levels of greater than 0.5 u/ml, allowed dialysis of 6 patients for 5 h and appreciably suppressed FPA generation during dialysis, with little effect on the KCCT.

It is concluded that the anti-factor Xa activity of Org 10172 may reflect its ability to inhibit fibrin during dialysis and that single bolus injection of Org 10172 may be a useful alternative method of achieving anticoagulation.
Identification of t-PA as the Major Active Plasminogen Activator in Human Milk
Authors: Marshall J M , Rees M C P , Cederholm-Williams S A
Published: July 2018

Human colostrum and milk contain high levels of plasminogen activator when compared with blood. These levels are inversely related to the duration of lactation and the activity appears to be due to tissue type plasminogen activator present in multiple (65-190 Kd) molecular weight forms.
Effects of a Thromboxane Synthetase Inhibitor and a cAMP Phosphodiesterase Inhibitor, Singly and in Combination, on Platelet Behaviour
Authors: Sills T , Heptinstall S
Published: July 2018

The effects of dazoxiben, a thromboxane synthetase inhibitor, and AH-P 719, a cAMP phosphodiesterase inhibitor, on arachidonic acid (AA)-induced platelet behaviour were determined. The levels of cAMP present in platelet-rich plasma (PRP) after stimulating the platelets with AA in the absence and presence of the agents were also measured.

AH-P 719, as well as dazoxiben, was more effective as an inhibitor of AA-induced platelet behaviour in PRP from some individuals than in PRP from others, and the effectiveness with which it inhibited platelet behaviour paralleled that of dazoxiben. A combination of both agents was more effective than either agent alone.

Both AH-P 719 and dazoxiben increased the level of cAMP in AA-stimulated platelets but again they were more effective in PRP from some individuals than others. A combination of AH-P 719 and dazoxiben always resulted in higher levels of cAMP than either agent alone.

These results imply that cAMP is involved in determining the effects of thromboxane synthetase inhibitors on platelet behaviour, and indicate that the anti-thrombotic potential of a combination of a thromboxane synthetase inhibitor and a cAMP phosphodiesterase inhibitor may be greater than that of the individual agents.
Fibrinolytic Activity and Protein C in Preeclampsia
Authors: Aznar J , Gilabert J , Estellés A , España F
Published: July 2018

Various parameters of the fibrinolytic system and antigenic and functional protein C and its inhibitor were studied during normal pregnancy and in patients with preeclampsia.

The fast acting tissue-type plasminogen activator inhibitor level was found to increase progressively during normal pregnancy. This increase was more evident in cases of severe preeclampsia (p<0.05). No variations were observed in protein C levels in normal pregnancies but a reduction in protein C level was noted in patients with severe preeclampsia (p<0.01). In preeclampsia, the protein C inhibitor level was higher than in normal pregnancy; it was also higher in normal pregnancy when compared to the control group.
Increase in Plasma Concentrationof Plasminogen Activator Inhibitor, Fibrinogen, von Willebrand Factor, Factor VIII: Cand in Erythrocyte Sedimentation Rate with Age
Authors: Aillaud M F , Pignol F , Alessi M C , Harle J R , Escande M , Mongin M , Juhan-Vague I
Published: July 2018

Elderly patients have previously been shown to have an increased plasma concentration of tissue plasminogen activator (t-PA) antigen (t-PAAg). Since the concentration of t-PA Ag dependson both free t-PA and t-PA complexed with inhibitors, mainly plasminogen activator inhibitor (PA inhibitor), we have investigated the relationship between the plasma concentration of PA inhibitor and age in 20 elderly and 20 young individuals. Elderly individuals showed a slight increase in PA inhibitor, in parallel with increase on others, acute-phase proteins, fibrinogen, von Willebrand factor, factor VIII :C, and the erythrocyte sedimentation rate. The increase i PA inhibitor as well as other acute-phase proteins in the elderly may be significant in relation to the increased incidence of thrombotic disease.
A Low Molecular Weight Heparin Alters the Fetal Coagulation System in the Pregnant Sheep
Authors: Andrew M , Ofosu F , Fernandez F , Jefferies A , Hirsh J , Mitchell L , Buchanan M R
Published: July 2018

Standard heparin and a LMWH, CY222 do not cross the placenta nor alter fetal coagulation when injected into the pregnant ewe. We found that another LMWH, Pharmuka-10169 (PK-10169) alters fetal coagulation without crossing the placenta in the pregnant sheep. To characterize this anticoagulant we measured the in vitro and in vivo effects of 125I-PK-10169 in maternal and fetal plasmas following administration of PK-10169 to the mother or fetus. The fetal anticoagulant activity was not neutralizable by protamine sulphate and was attributable to the inhibition of thrombin but not factor Xa. In vitro, the fetal anticoagulant activity had properties similar to dermatan sulphate : both catalyzed the inhibition of thrombin but not factor Xa by sheep plasma; and neither was neutralizable by protamine sulphate. These effects were due to the enhanced neutralization of thrombin by heparin cofactor II. We conclude that PK-10169 does not cross the placenta, but does induce the release of an endogenous dermatan sulphate-like substance which alters fetal coagulation.
Platelet Behaviour in Non-Insulin-Dependent Diabetes -Influence of Vascular Complications, Treatment and Metabolic Control
Authors: Peacock I , Hawkins M , Heptinstall S
Published: July 2018

Platelet-rich plasma was prepared from 47 patients with noninsulin-dependent diabetes treated with glibenclamide and metformin, and 21 controls. The release of radio-labelled 5-hydroxy-tryptamine in response to aggregating agents (adenosine diphosphate, adrenaline and sodium arachidonate), and the effects on release of a selective thromboxane inhibitor (UK-34787) were investigated. Subsequently, 20 of the diabetic subjects were chosen at random for treatment with insulin; the remainder continued to take tablets. Platelet studies were then repeated, in all patients, after 4 and 6 months.

The results showed an association between platelet behaviour and the presence of vascular complications, and were consistent with previous observations of reduced platelet reactivity in patients taking sulphonylureas. There was no correlation of platelet reactivity with blood glucose, glycosylated haemoglobin or lipid levels.
Differences in Coagulation and Haemostatic Parameters in Normal Women of Childbearing Age from Different Ethnic Groups and Geographical Locations
Authors: Bocaz J A , Barja P , Bonnar J , Daly L , Carrol A , Coutinho E , Goncalves M , Tsakok M , Koh S , Thomson J M , Poller L , Heady A , Holck S , Pinol A
Published: July 2018

A comparative study of coagulation and fibrinolytic laboratory parameters was undertaken in four countries (Salvador, Brazil; Singapore; Santiago, Chile and Dublin, Ireland) among apparently healthy women of reproductive age. A continuous external quality control scheme of the laboratory measurements was employed to permit comparison among centres. Significant and consistent differences were found between the four centres. In Dublin, the prothrombin time and activated partial thromboplastin time (APTT) were accelerated, and the specific factor assays showed more activity, whereas the antiprotease levels were higher than in the other centres. In Salvador, a contrasting tendency was found with longer prothrombin times and APTT and lower Factor VII and antiprotease levels. The results from the other two centres were approximately midway between these two extremes. The study has revealed important differences in the coagulation and haemostatic tests between women from widely diverse geographical areas. It is not certain whether these are due to ethnic, nutritional or economic factors but they may be related to the apparent varying incidence of thrombosis in these ethnic groups.
Low Molecular Weight Heparin (KABI2165) as Thromboprophylaxis in Elective Visceral Surgery
Authors: Koller Matthias , Schoch Ursula , Buchmann Peter , Largiadèr Felix , von Felten Arthur , Frick Paul G
Published: July 2018

In two randomized double-blind studies perioperative bleeding complications and thromboembolic events were assessed in 189 patients (pts) undergoing elective visceral surgery after subcutaneous administration of a low molecular weight (LMW) heparin fragment (KABI fragment 2165) or unfractionated (UF) heparin. The first study comparing 1 x 7′500 anti-factor Xa IU LMW heparin daily with 2 x 5′000 IU UF heparin was interrupted because of excessive bleeding complications (LMW heparin: 11/23 pts, UF heparin: 2/20 pts, p <0.01). In the second study (146 pts) the dose of LMW heparin was reduced to 1 x 2′500 anti-factor Xa IU. Bleeding complications (LMW heparin: 14.9%, UF heparin: 15.3%) and thromboembolic events (LMW heparin: 2.86%, UF heparin: 2.94%) were equal among the two groups.

2′500 anti-factor Xa IU/day of this LMW heparin fragment, corresponding to 15 mg/day, is the lowest dose of a LMW heparin used in a randomized clinical trial and was found to be a safe and efficient regimen in perioperative thrombosis prophylaxis. An advantage of LMW heparin over UF heparin is its once daily administration.
Interleukin 1 Stimulates Endothelial Cell Tissue Factor Production and Expression by a Prostaglandin-Independent Mechanism
Authors: Schorer Anna E , Kaplan Manuel E , Rao Gundu H R , Moldow Charles F
Published: July 2018

Activation of coagulation occurs at inflammatory sites following the ingress of mononuclear cells, and may result from alterations in the vessel wall. Since the monokine, interleukin 1, initiates diverse responses to inflammation, its ability to enhance vascular procoagulant activity was studied. Interleukin 1-treated cultured human endothelial cells acquired elevated levels of the procoagulant, tissue factor. This required de novo protein synthesis, was maximal at 2 h after exposure to interleukin 1, and resulted in persistently elevated cellular procoagulant activity. Tissue factor was later expressed (6-24 h) on the surface of uninjured endothelial cells. Endothelial cell procoagulant production and expression in response to interleukin 1 could be dissociated from endogenous prostaglandin metabolism, being insensitive to hydrocortisone, indomethacin, eicosatetrayionic acid and exogenous arachidonic acid. In addition, no increase in prostaglandin synthesis occurred during the interval in which tissue factor was synthesized. We therefore conclude that interleukin 1 stimulates endothelial synthesis and surface expression of tissue factor by a prostaglandin-independent mechanism.
The Interaction of the Factor Vlll/von Willebrand Factor Complex with Hematin
Authors: Green David , Furby Fiona H , Berndt Michael C
Published: July 2018

Intravenous infusion of hematin, used in the treatment of acute porphyria, induces a decline in the plasma factor VIH/von Willebrand factor complex (VIII/vWF) and thrombocytopenia. We investigated this problem by studying the interaction between hematin, purified VIII/vWF, and platelets in vitro. Hematin was labeled with either 59Fe or 3H and characterized by gel chromatography. Hematin self-aggregated, forming a complex with an average molecular weight of approximately 10,000 daltons. When incubated with VIII/vWF for 30 min at 37° C and applied to Sepharose CL-4B, the hematin eluted with the VIII/vWF in the void volume. Hematin inhibited the dissociation of factor VIII antigen (VIII :Ag) from the von Willebrand antigen (vWF:Ag) in 0.25 M CaCl2, and reversed the aggregation of VIII:Ag induced by 0.1 M 6-aminocaproic acid. Both hematin and the hematin-VIII/vWF complex bound to washed normal platelets and to platelets from a patient with Bemard-Soulier syndrome. Thromb-asthenic platelets were not aggregatable by hematin, and bound significantly less hematin-VIII/vWF than normal platelets suggesting that hematin-induced platelet activation was required for binding. Likewise, binding was inhibited by PGE1 which also prevented aggregation. We conclude that hematin forms complexes with VIII/vWF, alters the functional activity and dissociation of this compound, and participates in the binding of VIII/vWF to platelets.
Comparative Study of Size, Total Protein, Fibrinogen and 5-HT Content of Human and Canine Platelet Density Subpopulations
Authors: Mezzano Diego , Aranda Eduardo , Foradori Arnaldo
Published: July 2018

The size, total protein, fibrinogen and 5-HT content were evaluated in density subpopulations of human and canine platelets fractionated in linear arabinogalactan gradients. The methodology was assessed to ascertain that platelet separation was by density and to discard artifactual changes and platelet release during the procedure. EDTA or PGEi increased the size of human PRP-platelets, but not of dog platelets. In humans, high density (HD) platelets were 1.26 times larger and contained 1.88 times more fibrinogen, 2.23 times more 5-HT and 1.37 times more protein than low density (LD) platelets; in dogs, these density cohorts did not differ in protein content, but LD platelets were 1.29 times larger and had 1.33 times more fibrinogen and 5-HT than HD platelets. These findings suggest that cell density is mostly dependent on the protein content per unit volume of platelets (and not on dense bodies). The differences in fibrinogen and 5-HT content between HD and LD cohorts in humans and dogs may be related to platelet age. The difference in volume between HD and LD platelets in dogs is of uncertain interpretation.
Verapamil Inhibits Platelet Aggregation by a Calcium-Independent Mechanism
Authors: Bonadonna Giovanni , Lechi Clara , Corradini Paola , Sinigaglia Daniela , De Togni Piero , Miroslawa Grzeskowiak
Published: July 2018

We studied the inhibitory effects of the calcium channel blocker verapamil both on platelet aggregation and intracellular calcium [Ca2+]i in platelets loaded with a fluorescent indicator (quin 2).

The inhibitory effects of verapamil on the platelet aggregation response to both thrombin and ionomycin were seen to be clearly dissociated from the verapamil-induced inhibition of the [Ca2+]i increase produced by these agonists. Verapamil-induced inhibition of platelet aggregation was also obtained when using the “calcium-independent” agonist phorbol-myristate acetate (PMA). It may be deduced that a calcium-independent mechanism plays a role in verapamil-induced inhibition of platelet aggregation. We postulate that this mechanism may operate via a protein-kinase C pathway.
Intrathoracic Platelet Accumulation in the Guinea-Pig Induced by Intravenous Administration of Arachidonic Acid - Effect of Cyclooxygenase and Thromboxane Synthetase Inhibitors
Authors: Barrett P A , Butler K D , Shand R A , Wallis R B
Published: July 2018

Intravenous administration of arachidonic acid to guinea-pigs caused a dose-related, rapid accumulation of 51Cr-labelled platelets in the thorax. Inhibitors of cyclooxygenase inhibited the platelet accumulation, induced by arachidonic acid (30 mg/kg), at doses which did not alter the thoracic blood volume (as measured by 131I-labelled human albumin). Thromboxane synthetase inhibitors had different effects on platelet accumulation depending on the dose. CGS 12970 (3 mg/kg) and N(1-carboxyheptyl) imidazole (100 mg/kg) reduced platelet accumulation. High doses of CGS 12970 and CGS 13080 caused an apparent enhancement of platelet accumulation which was associated with pooling of blood in the thorax, as measured by either 131I-labelled human albumin or 51Cr-labelled erythrocytes. This increase in thoracic blood volume was abolished if the guinea-pigs were also pretreated with diclofenac (1 mg/kg) in addition to the thromboxane synthetase inhibitor. Increases in thoracic blood volume were also obtained following infusions of PGI2 but not PGD2 or PGE2.
Different Effects of Aspirin, Dipyridamole and UD-CG 115 on Platelet Activation in a Model of Vascular Injury: Studies with Extracellular Matrix Covered with Endothelial Cells
Authors: Eldor A , Vlodavsky I , Fuks Z , Muller T H , Eisert W G
Published: July 2018

Cultured endothelial cells produce an extracellular matrix (ECM) which activates platelets, similarly to deendothelialized vascular segments. Platelet-rich plasma (PRP) was incubated with endothelial cells cultures seeded in various densities on ECM. The interaction of the platelets with this artifical intima was evaluated by phase microscopy and by thromboxane A2 (TXA2) and prostacyclin (PGI2) measurement. Large platelet aggregates were formed on exposed ECM. Platelets aggregation but not adhesion on the ECM was markedly inhibited by the presence of endothelial cells. Pretreatment of the endothelial cells with 0.1 mM aspirin reduced their PGI2 synthesis and was associated with platelet aggregation on the ECM. 10 μM dipyridamole markedly inhibited platelet activation by ECM when the drug was added to citrated whole blood before PRP preparation. UD-CG 115 which elevates cyclic AMP in cardiac muscle, inhibited platelet aggregation and TXA2 production induced by ECM, in the presence as well as in the absence of endothelial cells, without any effect on endothelial PGI2 production.
Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding
Authors: Tripodi A , Krachmalnicoff A , Mannucci P M
Published: July 2018

Four members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.
The Plasmin Heavy Chain - Urokinase Conjugate: A Specific Thrombolytic Agent
Authors: Nakayama Y , Shinohara M , Tani T , Kawaguchi T , Furuta T , Izawa T , Kaise H , Miyazaki W , Nakano Y
Published: July 2018

Low molecular weight urokinase (LMW-UK) was coupled to the heavy chain of plasmin to make it able to bind to fibrin. The purified conjugate (PHC-UK conjugate), which consisted of equimolar concentrations of each starting material had a molecular weight of 93,600, bound tightly to fibrin-monomer-Sepharose and was not washed off with 1 M NaCl, but was eluted specifically with s-amino caproic acid. The conjugate showed higher fibrinolytic activity than HMW-UK. A control conjugate prepared by coupling human serum albumin to LMW-UK (HSA-UK conjugate) showed the same fibrinolytic activity as HMW-UK. The half-lives of these two conjugates in rabbits were about 3 times that of HMW-UK. In an experimental pulmonary embolism model in rabbits, the PHC-UK conjugate showed about 10 times higher thrombolytic activity than HMW-UK, while the HSA-UK conjugate showed similar thrombolytic activity as HMW-UK, and moreover caused severe systemic fibrinogen breakdown. Thus the significant increase in thrombolytic activity after injection of PHC-UK conjugate into rabbits may be due to its newly acquired fibrin binding activity, and not to increase in its half-life. It is concluded that the PHC-UK conjugate may be useful in treatment of thrombosis.
The Negative Impact of Biological Variation in the Effect and Clearance of Warfarin on Methods for Prediction of Dose Requirements
Authors: Weiss Peretz , Halkin Hillel , Almog Shlomo
Published: July 2018

Within-individual variation over time in the clearance (Cl) and effect (PT%) of warfarin, was measured in 25 inpatients (group I) studied after standard single or individualized split loading doses and 1-3 times (n = 16) 8-16 weeks later during maintenance. Mean Cl (2.5 α 0.9 ml/min) was similar in both phases but significant changes occurred in 6/16 patients, exceeding those expected from within-individual variation alone (defined by its 95% tolerance limits -24% to +62%). Initial PT% (21 α 5) was unaffected by dosing schedule, total or free plasma warfarin, varying between patients by only 18-24%. Mean initial and maintenance dose-PT% ratios (8.2 mg/d: 21% and 4.1 mg/d: 40%) were similar but significant changes in sensitivity to warfarin occurred in 4/16 patients. In group I and 64 other outpatients on maintenance therapy, between-individual variability was 36-52% for Cl and 49-56% for effect. PT% correlated best (r = 0.56) with free and total plasma warfarin but poorly with dose (r = 0.29), with only 30% of PT% variance explained at best, due to high between patient variability.

Warfarin dose prediction whether based on extrapolation from initial effects to the maintenance phase, or on iterative methods not allowing for between- or within-patient variation in warfarin clearance or effect which may occur independently over time, have not improved on empirical therapy. This, due to the elements of biological variability as well as the intricacy of the warfarin - prothrombin complex interaction not captured by any kinetic-dynamic model used for prediction to date.
Comparison of Tryptic Fragments of von Willebrand Factor Involved in Binding to Thrombin-Activated Platelets with Fragments Involved in Ristocetin-Induced Binding and Binding to Collagen
Authors: Houdijk Wim P M , Girma Jen-pierre , van Mourik Jan A , Sixma Jan J , Meyer Dominique
Published: July 2018

Previously we have studied the binding domains on von Willebrand factor (vWF) involved in ristocetin-induced binding to platelets (ristocetin binding domain, RBD) and in the binding of vWF to collagen (collagen binding domain, CBD) using tryptic fragments of 125I-labelled vWF (21, 23). We have also reported on the RBD, CBD and the domain on vWF involved in the binding to thrombin activated platelets (thrombin binding domain, TBD) using vWF-fragments prepared by digestion with staphylococcal protease V8 (25).

In the present study, we have digested 125I-vWF with TPCK-trypsin and we have performed at various times of digestion immuno-precipitation with Mab 9, the antibody inhibiting binding of vWF to thrombin activated platelets. The data were compared with the immunoprecipitation patterns simultaneously obtained with CLB-RAg 35 which inhibits binding of vWF in the presence of ristocetin and with CLB-RAg 201, which inhibits binding of vWF to collagen. At 90 min, Mab 9 and CLB-RAg 201 precipitated similar high molecular weight bands, whereas CLB-RAg 35 precipitated bands at 180 and 120 kDa. After 24 h, Mab 9 precipitated bands at 200, 155, 116 and 85 kDa; CLB-RAg 201 precipitated a band at 48 kDa and CLB-RAg 35 a band at 116 kDa. Two-dimensional electrophoresis demonstrated that the high molecular weight bands, precipitated by Mab 9 and CLB-RAg 201 at 90 min, were identical. The 116 kDa fragment recognized by CLB-RAg 35 had a different subunit composition than the 116 kDa fragment precipitated by Mab 9.

These data indicate that there is an early split in the vWF subunit which yields a large fragment, containing the CBD and the TBD, and a smaller fragment with the RBD. Later, there is a split in the large fragment and the CBD and the TBD are separated. Comparison with data obtained with Staph protease V8 indicate that the CBD is located between the RBD and TBD.
Differential Detection of Single-Chain and Two-Chain Urokinase-Type Plasminogen Activator by a New Immunoadsorbent-Amidolytic Assay (IAA)
Authors: Corti Angelo , Luisa Nolli Maria , Cassani Giovanni
Published: July 2018

A new immunoadsorbent-amidolytic assay (IAA) for the specific differential detection of two-chain urokinase-type plasminogen activator (tcu-PA) and its single-chain precursor (scu-PA) in cell culture supernatants has been developed. The assay combines the selectivity of immunoassays with the specificity of enzyme activity assays exploiting both the antigenic and enzymatic properties of the two proteins. tcu-PA and scu-PA are selectively immunoadsorbed on the wells of a microtiterplate coated with the monoclonal antibody 5B4 and tested for enzymatic activity before and after activation by plasmin treatment. Both proteins are determined with similar efficiency since overlapping dose-response curves were obtained in the range between 12.5-200 ng/ml. The assay has been used to determine tcu-PA and scu-PA in A431 human epidermoid carcinoma cell supernatants. The analytical recoveries for tcu-PA and scu-PA added to A431 cell supernatants were 95.2% and 96.9% respectively. The intra- and inter-assay variations (CV) were 5.5% and 9.0% for tcu-PA and 9.7% and 9.8% for scu-PA respectively.
Effect of the Calcium-Entry Blocking Agent Nifedipine on Activation of Human Platelets and Comparison with Verapamil
Authors: Han P , Boatwright C , Ardlie N G
Published: July 2018

The effects of the calcium-entry blocking agent nifedipine on the activation of human platelets by various agonists has been studied and compared with verapamil. Like verapamil, nifedipine inhibited platelet aggregation and secretion caused by collagen, the second phase of ADP-induced aggregation, and aggregation caused by the ionophore A23187. Both agents inhibited the formation of TXB2 from endogenous arachidonate, whereas only nifedipine inhibited platelet aggregation and decreased TXB2 formation caused by exogenous arachidonate without inhibiting uptake. These results indicate that both calcium-blocking agents may be inhibiting the release of arachidonate in platelets by phospholipases, and that nifedipine also inhibits the formation and action of thromboxane A2 in platelets. Epinephrine-induced aggregation was inhibited by low concentrations of verapamil while nifedipine only inhibited aggregation by epinephrine at much higher concentrations. It is suggested that low concentrations of verapamil inhibit epinephrine-induced aggregation by interacting with platelet α-adrenergic receptors, and that higher concentrations of both calcium-blocking agents inhibit platelet responses to other aggregating agents by preventing intracellular calcium mobilization.
Plasma Fibrinopeptide A and Beta-Thromboglobulin in Patients with Chest Pain
Authors: Douglas J T , Lowe G D O , Forbes C D , Prentice C R M
Published: July 2018

Plasma levels of β-thromboglobulin (BTG) and fibrinopeptide A (FPA), markers of platelet release and thrombin generation respectively, were measured in 48 patients within 3 days of admission to hospital for acute chest pain. Twenty-one patients had a confirmed myocardial infarction (MI); 15 had unstable angina without infarction; and 12 had chest pain due to noncardiac causes. FPA and BTG were also measured in 23 control hospital patients of similar age. Mean plasma BTG levels were not significantly different in the 4 groups. Mean plasma FPA levels were significantly higher in all 3 groups with acute chest pain when compared to the control subjects (p < 0.01), but there were no significant differences between the 3 groups. Increased FPA levels in patients with acute chest pain are not specific for myocardial infarction, nor for ischaemic chest pain.
Dissociation of Factor VIII Procoagulant Antigen VIII : CAg and Factor VIII Related Antigen VIIIR : Ag by EDTA - Influence of Divalent Cation on the Binding of VIII: CAg and VIIIR :Ag
Authors: Tran T H , Duckert F
Published: July 2018

Assuming 1 U/ml in titrated plasma, the VIII: CAg concentration was found 1.66 U/ml in EDTA-plasma, 1.09 U/ml in heparinized plasma and 0.67 U/ml in serum. Addition of 10 mmol/1 EDTA to titrated and heparinized plasmas increased VIII: CAg 1.5fold. There was no increase of VIII: CAg in serum. Gel filtration of plasmas on different anticoagulants showed an elution of VIII: CAg in the void volume Vo and in the later fractions. The VIII: CAg amount detected in the internal volume increased following the series heparin < citrate < EDTA. Serum VIII: CAg was eluted at 2.2 Vo. Presence of EDTA in the elution buffer or incubation of plasma with EDTA prior to chromatography caused a displacement of practically all VIII: CAg amount in the internal volume with a peak at 2.2-2.3 Vo. VIIIR: Ag was exclusively detected in the void volume.

Removal of divalent cation by chelation likely exposes more antigenic determinants of VIII: CAg, which are otherwise masked by steric hindrance due to VIIIR: Ag in citrated and heparinized milieu. Moreover gel filtration of plasma in the presence of EDTA completely dissociates VIII: CAg from VIIIR :Ag. The VIII: CAg fragment, having an estimated molecular weight of 70,000, might also be present in serum.
Determination of Factor XIII Activity and of Factor XIII Inhibitors Using an Ammonium-Sensitive Electrode
Authors: Hellstern P , Schilz K , von Blohn G , Wenzel E
Published: July 2018

An assay for rapid factor XIII activity measurement has been developed based on the determination of the ammonium released during fibrin stabilization. Factor XIII was activated by thrombin and calcium. Ammonium was measured by an ammonium-sensitive electrode. It was demonstrated that the assay procedure yields accurate and precise results and that factor XIII-catalyzed fibrin stabilization can be measured kinetically. The amount of ammonium released during the first 90 min of fibrin stabilization was found to be 7.8 ± 0.5 moles per mole fibrinogen, which is in agreement with the findings of other authors. In 15 normal subjects and in 15 patients suffering from diseases with suspected factor XIII deficiency there was a satisfactory correlation between the results obtained by the “ammonium-release-method”, Bohn’s method, and the immunological assay (r1 = 0.65; r2= 0.70; p<0.01). In 3 of 5 patients with paraproteinemias the values of factor XIII activity determined by the ammonium-release method were markedly lower than those estimated by the other methods. It could be shown that inhibitor mechanisms were responsible for these discrepancies.
New “Ex Vivo” Radioisotopic Method of Quantitation of Platelet Deposition – Studies in Four Animal Species
Authors: Badimon L , Fuster V , Chesebro J H , Dewanjee M K
Published: July 2018

We have developed a sensitive and quantitative method of “ex vivo” evaluation of platelet deposition on collagen strips, from rabbit Achilles tendon, superfused by flowing blood and applied it to four animal species, cat, rabbit, dog and pig. Autologous platelets were labeled with indium-111-tropolone, injected to the animal 24 hr before the superfusion and the number of deposited platelets was quantitated from the tendon gamma-radiation and the blood platelet count.

We detected some platelet consumption with superfusion time when blood was reinfused entering the contralateral jugular vein after collagen contact but not if blood was discarded after the contact. Therefore, in order to have a more physiological animal model we decided to discard blood after superfusion of the tendon.

In all species except for the cat there was a linear relationship between increase of platelet on the tendon and time of exposure to blood superfusion. The highest number of platelets deposited on the collagen was found in cats, the lowest in dogs.

Ultrastructural analysis showed the platelets were deposited as aggregates after only 5 min of superfusion.
The Stability of Tissue Plasminogen Activator (t-PA)
Authors: Gaffney P J , Templeman J , Curtis A D , Campbell P J
Published: July 2018

Since tissue-type plasminogen activator (t-PA) derived from tissue culture and recombinant DNA procedures has been proposed for use in thrombolytic therapy in man, it is essential that
the t-PA molecule should display reasonable stability in a lyophilised state to facilitate its usefulness. In this study, four laboratories compared the potencies of three preparations of t-PA following storage at 4°, 20°, 37° and 45° C, using each t-PA stored at -20° C as a reference (100% activity) in each case. A pig heart extract of t-PA was the most stable, losing no activity when stored for 30 days at 37° C, while two melanoma cell tissue culture extracts varied in their storage behaviour. One was quite stable at 37° C (losing about 3% of its activity) while the other lost about 16%. Thus both the pig heart t-PA and one t-PA from melanoma cell culture proved suitable for the further development of reference standards for t-PA activity.
Anticoagulant Properties of Three Mucopolysaccharides Used in Rheumatology
Authors: Bauer F , Schulz P , Reber G , Bouvier C A
Published: July 2018

Three mucopolysaccharides (MPS) used in the treatment of degenerative joint disease were compared to heparin to establish their relative potencies on 3 coagulation tests, the aPTT, the antifactor X a activity and the dilute thrombin time. One of the compounds, Arteparon®, was one fourth as potent as heparin on the aPTT, but had little or no influence on the 2 other tests. Further in vitro studies suggested that Arteparon® acted at a higher level than factor Xa generation in the intrinsic amplification system and that its effect was independent of antithrombin III. In vivo administration of Arteparon® confirmed its anticoagulant properties, which raises the question of the clinical use of this MPS.
Systemic Fibrinolytic Activity and Inhibitor Levels During Treatment of Deep Vein Thrombosis with Urokinase and Streptokinase
Authors: Theiss W , Asbeck F , Kriessmann A , Trübestein G , Knoch K , de Swart C A M , Marbet G A , van de Loo J C W
Published: July 2018

In a prospective, randomized trial 33 patients with deep vein thrombosis were treated either with 2,200 or 1,100 IU/kg/h urokinase or with 100,000 IU/h streptokinase for at least 6 days. While streptokinase was given continuously, urokinase was administered intermittently (12 hr urokinase alternating with 12 hr heparin).

Urokinase treatment resulted in a dose-dependent fibrinolytic state with shortening of the euglobulin clot lysis time, easily demonstrable amidolytic activity and moderate decrease of plasminogen. At the end of each urokinase-free interval the fibrinolytic activity had mostly faded, but was reproducibly elicited again by each new urokinase administration. Streptokinase immediately evoked the customary, intense fibrinolytic state, which progressively tapered off as plasminogen fell to 1% of its pretreatment concentration. In all treatment groups α-2-antiplasmin dropped to approximately 40% of its initial value during the first 12 hr with a further decrease to about 20% after 6 days, α-2-macroglo- bulin fell only moderately with either urokinase regimen, whereas it decreased progressively to 45% under streptokinase.

While the fibrinolytic activity decreased under streptokinase over the 6-day infusion period, it appeared to increase with each successive urokinase infusion particularly with 1100 IU/kg/h. Thus the final euglobulin clot lysis times and the final fibrinogen concentrations were similar in all three treatment groups on the sixth day.
The Effect of Two Low Doses of Aspirin on Whole Blood Thromboxane and Prostacyclin Generation in Healthy Subjects
Authors: Davì G , Custro N , Novo S , Mattina A , Strano A
Published: July 2018

The effects of two low doses of aspirin (20 mg and 100 mg) on prostacyclin and thromboxane formation during whole blood clotting were studied in 8 healthy volunteers.

A single 100 mg aspirin dose caused more than 90% reduction of both serum TXB2 and 6-keto-PGF1α; a single 20 mg dose of aspirin inhibited serum TXB2 more than 6-keto-PGF1α but effects on these two products could not be completely dissociated.

However, the effect of a single 20 mg aspirin dose on serum TXB2, was of much longer duration than its inhibitory effect on PGI2 synthesis during whole blood clotting.
Coagulant and Anticoagulant Actions of Australian Snake Venoms
Authors: Marshall Lorraine R , Herrmann Richard P
Published: July 2018

A systematic study was made of the action on the plasma coagulation system of 20 Australian and Papuan Elapid and Hydrophiid snake venoms and compared with 4 Crotalid venoms and 1 Viper. The majority of Australian venoms were shown to be prothrombin activators with variable dependence on the presence of factor V phospholipid and calcium. None of these venoms had strong thrombin like activity in contrast to the Crotalid venoms which were powerfully thrombin like. The Crotalid venoms were also strongly fibrinolytic unlike the Elapid venoms which showed no or minimal evidence of fibrinolytic activity.

Four Elapid venoms and 2 Crotalid venoms showed anticoagulant activity which contained neither antithrombin nor fibrinogenolytic activity and may act upon the prothrombin complex.
Rapid Quantitation of Plasma Heparin and Antithrombin III Levels for Cardiopulmonary Bypass Monitoring, Using Fluorometric Substrate Assays
Authors: Savidge G F , Kesteven P J , Al-Hasani S F , O’Brien P F
Published: July 2018

Plamsa heparin and AT III levels were determined using recently developed fluorometric substrate techniques on serial samples taken from 9 unselected patients undergoing open-heart surgery on cardiopulmonary bypass. The fluorometric assay for AT III and established clotting and immunological methods showed a highly significant correlation (p <0.001). Heparin recovery was reduced in all cases, and plasma levels (fluorometric assay) demonstrated poor correlation to Whole Blood Activated Clotting Time (Hemochron) estimations. AT III levels were dramatically reduced during bypass, and in 3 cases reached levels below 0.08 iu/ml. Heparin reversal schedules based upon empirical dosage led to excessive protamine administration by a mean factor of 3.3, as assessed by in vitro neutralization of standardized heparin concentrations.
The Effect of Polyunsaturated Fatty Acids on Endothelial Cells and Their Production of Prostacyclin, Thromboxane and Platelet Inhibitory Activity
Authors: Brox Jan H , Nordøy Arne
Published: July 2018

Primary cultures of human endothelial cell monolayers were incubated with albumin-bound fatty acids of the ω-3 and ω-6 families for a maximum of 24 hrs, to investigate the production of 6-keto-PGF1α, TXB2 and platelet inhibitory activity (PIA). Arachidonic acid was a potent stimulator of all parameters. The release of 6-keto-PGF1α was significantly reduced by equimolar concentrations of linoleic, dihomogamma linolenic and eicosapentaenoic acids, but not by linolenic acid. PIA was not similarity affected.

Dihomogamma linolenic add was also a weak stimulator of 6- keto-PGF1α and PIA, but reduced the content of both in the cells after 24 hrs. Eicosapentaenoic and docosahexaenoic acids both depressed 6-keto-PGF1α production but PIA was maintained after 24 hrs. Indomethacin always blocked 6-keto-PGF1α and PIA production. None of the effects correlated to release of 51CR from prelabelled cells.
A Sex Difference in the Effect of Aspirin on “Spontaneous” Platelet Aggregation in Whole Blood
Authors: Harrison M J G , Weisblatt E
Published: July 2018

Platelet aggregation can be measured in whole blood by monitoring the fall in single platelet count in an electronic platelet counter. The aggregation that occurs when whole blood is stirred in a small cuvette (“spontaneous aggregation”) or upon the addition of collagen has been studied in citrated whole blood from male and female volunteers. Aspirin 40 μg ml/1 inhibited aggregation induced by collagen in both sexes but spontaneous aggregation was only affected by aspirin in males. These results may help explain the sex difference apparent in the results of some clinical trials of aspirin as an antithrombotic agent.
Investigations of the Lupus-Like Inhibitor By-Passing Activity of Platelets
Authors: Howard Margaret A , Firkin Barry G
Published: July 2018

Plasmas from ten patients with a lupus or lupus-like inhibitor were investigated. In each case the partial thromboplastin time with kaolin (PTTK) was prolonged and failed to correct on the addition of an equal volume of normal plasma. Activated control platelets corrected the inhibitory effect in the PTTK or thrombin generation time (TGT) in every instance. Activated autologous platelets were as effective as control platelets and may thus explain why bleeding is rarely associated with the lupus inhibitor. Experiments using platelets or plasma from patients congenitally deficient in a single clotting factor or normal washed platelets resuspended in deficient plasma indicated that the inhibitor bypassing activity is platelet and not plasma derived. Platelet fractionation studies suggested that this activity is localised at the platelet membrane.
Cyanide Prevents the Inhibition of Platelet Aggregation by Nitroprusside, Hydroxylamine and Azide
Authors: Schwerin Francis T , Rosenstein Robert , Smith Roger P
Published: July 2018

Sodium cyanide (CN-) in concentrations of 10 uM or more prevented the inhibition of epinephrine (2.5 uM) and of ADP (4.0 uM) induced primary and secondary aggregation brought about by 10 uM sodium nitroprusside (SNP). Cyanide alone in the same concentration had no effect on platelet aggregation induced by epinephrine or ADP. Even when the addition of CN- was delayed for as long as 9 min after epinephrine and SNP, it immediately reversed the SNP block and initiated a bimodal wave of aggregation. The effect of CN- on SNP inhibition of platelet aggregation appears to be competitive and reversible. Although they are less potent inhibitors of platelet aggregation than SNP, the effects of hydroxylamine (HA) and azide were also prevented by SNP. In our hands, sodium nitrite did not inhibit platelet aggregation consistently. The inhibitory effects of glyceryl trinitrate, papaverine and nitric oxide hemoglobin on platelet aggregation were not prevented by CN-. These interactions probably have no significance in vivo, but they indicate that SNP, HA and azide act on platelets and on vascular smooth muscle by similar or identical biochemical mechanisms. They also suggest that there are at least two sub-classes of so-called nitric oxide vasodilators. The effect of CN- may be mediated through an inhibition of the formation of nitric oxide from SNP, HA and azide.