bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Platelet Activation In The Absence Of Aggregation: A Calcium-Dependent Phenomenon Independent Of Thromboxane Formation
Authors: Levy-Toledan S , Maclouf J , Bryon P A , dela Baume H , Savariau E , Hardisty R , Caen J P
Published: July 2018

In response to the ionophore A 23187, thrombasthenic platelets undergo a change in light transmission (LT) using either PRP or washed platelets. This change was accompanied by a normal release of 14C serotonin (5HT) and thromboxane (TX) synthesis in absence of aggregation; lactic dehydrogenase determinations showed no evidence of platelet lysis. Ultrastructural qualitative electron microscopy revealed the formation of the central gel mass and the central apposition of organelles but the platelets were loosely packed. This ultrastructural change which was accompanied by biochemical phenomena was quantified; this includes an increase of the elongation coefficient, a decrease of the circularity coefficient as well as a decrease of the percentage of granules and surface connecting system in comparison with the total volume of the cell.

The change in light transmission was not inhibited by non-steroidal anti-inflammatory drugs (aspirin, indometha- cin, flurbiprofen) suggesting a dissociation between a normal release of 14C-5HT and an absence of TX formation. Moreover it was not inhibited by creatine phosphate/ creatine phosphokinase, prostaglandin E1 or cytochalasine and/or colchicine; it was then not dependent on ADP, cAMP or the integrity of microfilaments and microtubules. However chlorpromazine, TMB8 and dibucaine, drugs which interfere with intracellular membrane transport of calcium ions inhibited this platelet activation (change in LT, 14C-5HT release or TX synthesis).

The stimulation of internal calcium fluxes by the ionophore was sufficient to induce platelet activation in the absence of aggregation; it was found to be independent of TX formation.
Involvement Of Calmodulin In Platelet Reaction
Authors: Kösaki G , kambayshi J , Ohno H , Morimoto K , Hakata H
Published: July 2018

Calmodulin is a ubiquitous Ca2+-binding protein, modulating a number of Ca2+-dependent cellular reactions. In blood platelets, involvement of calmodulin has been demonstrated in the activation of myosin light chain kinase and of phospholipase A2. In the present study, involvement of this protein in platelet reaction was thoroughly investigated, utilizing phenothiazines(chlorpromazine and trifluoperazine) known as selective inhibitors of calmodulin.

Phenothiazines in micromolar concentration completely inhibited platelet aggregation and secretion induced by ADP epinephrine, collagen, thrombin or Ca2+-ionophore. They also completely inhibited the aggregation triggered by arachido- nate or by the mixture of thromboxane A2 and prostaglandin endoperoxides, which is probably due to the inhibition of activation of myosin light chain kinase. As another possible involvemnt of calmodulin in the reaction, the effect of phenothiazines on the release of arachidonate from phospholipids was studied in stimulated platelets to which radioactive arachidonate had been incorporated. The release was dose-dependently inhibited by either of phenothiazines. Then, their effect on the hydrolysis of phosphatidylinositol by platelet sonicates was investigated, using 3H-myoinositol phosphatidylinositol as substrate. Either of phenothiazines suppressed the hydrolysis in the micromolar concentration, indicating their inhibition on phosphatidylinositol specific phospholipase C.

These observations suggest involvement of calmodulin in at least two steps of platelt reaction: one is the step of the release of arachidonate from phospholipids (the activation of phospholipase C in addition to phospholipase A2) and the other is the step of contraction of platelet actomyosin after the formation of thromboxane A2 (the activation of myosin light chain kinase).
Inhibition Of Platelet Adhesion To Collagen By Prostaglandins (PGI2 PGE1, PGD2) Its Possible Correlation With Camp/ Adenylate Cyclase System
Authors: Karniquian A G , Leqrand Y J , Caen J P
Published: July 2018

The purpose of this work Was to investigate if the inhibitory effect of Prostacyclin (PGI2) on the ex vivo platelet adhesion to rabbit subendothelium (under defined conditions at a shear rate of 860 sec-1) is explained by an inhibition of platelet adhesion to collagen. We therefore applied a sensitive method to measure the adhesion of lllIn labelled platelets to collagen ; this method has permitted to check the effect of very low concentrations of PGI2 and also of two other prostaglandins (PGs) which have been shown to inhibit platelet aggregation : PGE1 and PGD2, An inhibition of adhesion has been observed which was short lasting with PGE1, and of longer duration (more than 5 minutes) with PGI2 and PGD2 : the maximum inhibition using 1 µM of each compound was 92 % for PGI2, 69 % for PGD2 and 59 % for PGE1 This inhibition was going in parallel with platelet cAMP increase and ceased when the cAMP decreased. Theophylline (10-3M), a phosphodiesterase inhibitor, potentiated the inhibitory effect of PGE1 on platelet adhesion. The addition of exogenous N6- O2’ dibutyryl cAMP (10-5 - 10-3M) reproduces the inhibitory effect of PGs on platelet adhesion, suggesting that PGs act on platelet adhesion to collagen via adenylate cyclase/cAMP system. Prostaglandins caused an inhibition of collagen induced platelet release, this inhibition was the same, wether it was measured on the α granules ( βthromboglobulin) or dense bodies (ADP and 5HT) content. The dose of prostaglandins which provokes a 50 % inhibition (ID50) is higher for the release : (PGI2 ID50 :βTG = 7.2 10-11m, 5HT = 7 10-11m, PGE1 ID50βTG = 10-9M, 5HT = 0.6 10-9M, PGD2 ID50 :βTG = 1.2 10-9M, 5HT = 1.6 10-9m) than for platelet adhesion (PGI2 ID50 =
7 10-lOM, PGE1 ID50 = 10-8M, PGD2 ID50 = 7.5 10-7M.
Effect Of TicloPidine On Platelet Deposition In Gore-Tex And Autologous Vein Grafts
Authors: Murphy K P , Dewanjee M K , Fuster V , Didisheim P , Kaye M P
Published: July 2018

To quantitatively study platelet deposition on arterial prostheses and the effect of platelet inhibitory therapy with ticlopidine, 14 dogs underwent bilateral femoral artery excision and replacement with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Five dogs received 60 mg/kg/day of ticlopidine orally on four consecutive days prior to surgery. Nine untreated dogs served as controls. Collagen-induced platelet aggregometry studies were performed at the end of day 4. Autologous indium-111-labeled platelets were injected 24 hours prior to surgery to serve as aplatelet marker. All animals received heparin (1 mg/kg I.V.) 5 minutes prior to excision of the arterial segment. Grafts were removed 1 hour following resumption of blood flow and gently flushed with 30 cc normal saline. Radioactivity per unit weight of Gore-Tex, vein and blood was determined with a gamma counter. The relative radioactivity with respect to the internal reference standard, blood, is tabulated below.

Ticlopidine treated dogs showed a 15-fold reduction in platelet deposition on Gore-Tex grafts and a 3-fold reduction on venous grafts. Platelet deposition was reduced significantly in Gore-Tex grafts (P<0.001) to a level comparable to that in autologous veins. Platelet inhibitory effect was also observed in collagen-induced platelet aggregometry.

According to this “in vivo” data, ticlopidine appears to be a promising platelet inhibiting agent.
The Polymerization Of Fibrin And The Clearance Of Fibrinopeptide B By Thrombin
Authors: Hurlet-Jensen A , Cummins H Z , Nossel H L , Liu C Y
Published: July 2018

Thrombin cleaves fibrinogen to release fibrinopeptide A (FPA) and B (FPB) to form fibrin I and II respectively. Initially FPA is cleaved more rapidly than FPB but later the FPB cleavage rate increases and is similar to that of FPA. Studies were made at 1.65 μM fibrinogen concentration, 0.02U thrombin/ml, pH 7.4, 0.15M NaCl, 37°. The initial FPB cleavage rate was 7 pmol/min and then increased to 70 pmol/min. The accelerated rate of cleavage was associated with fibrin polymerization as indicated by light scattering and absorbance measurements. Fibrin polymerization was inhibited by the synthetic tetrapeptide Gly-Pro-Arg-Pro. At a concentration of tetrapeptide of 23mM (1400 fold molar ratio to fibrinogen) no detectable polymerization occurred over 180 min. At this tetrapeptide concentration the FPA cleavage rate was unaltered from that occurring without tetrapeptide but the rate of FPB cleavage did not show the acceleration normally associated with polymerization although virtually all the FPB was cleaved. The cleavage rates of FPB from fibrin I polymer and monomer were then compared. Fibrin I polymer was prepared by treating fibrinogen with reptilase and fibrin I monomer by similar treatment in the presence of excess tetrapeptide. The cleavage rate from monomer was similar to the initial slow cleavage rate when thrombin was added to fibrinogen. The cleavage rate from polymer was 25 fold more rapid than that from monomer. These data indicate that thrombin cleaves FPB slowly but completely from fibrin I monomer and at least 25-fold more rapidly from polymer. The data do not establish that thrombin cleaves FPB from fibrinogen.
In Vivo Evaluation Of The Antithrombotic Effects Of Some Polysaccharides Of Ultra Low Molecular Weight
Authors: Fareed J , Messmore H L , Choay J , Lormeau C , Petitou M , Andersen A , Larsen C H , Stulc J
Published: July 2018

Ultra low molecular weight saccharide fragments (ULMFs) have been obtained from porcine mucosal heparin (PMH) by extraction (e-ULMF) and by bacterial heparinase depolymerization (h-ULMF-8) processes. Both fragments showed a strong anti Xa activity (>2000 u/mg units, Yin and Wessler, J. Lab. Clin. Med. 81, 298, 1973) and possess relatively weak potencies in the US Pharmacoepial (<40 USP u/mg) and other conventional coagulant assays (activated and non activated partial thromboplastin time, thrombin time and whole blood activated recalcification times). Since ULMFs showed a strong anti Xa activity, we evaluated their antithrombotic actions in a modified stasis-thrombosis model (Wessler et. al. J. App. Phys. 14, 943, 1959) challenging the animals with various thrombogenic stimuli; activated and non activated prothrombin complex concentrates, factor Xa concentrates and human serum. h-ULMF-8 at dosage <0.125 mg/kg (<250 Anti Xa u/kg) IV and <1.0 mg/kg (>2000 anti Xa u/kg) SC completely protected the thrombogenic effects of various thrombogenic agents, whereas PMH at these dosages failed to produce any protection in pre and post treatment regiments. Similar studies with e-ULMF showed protection, however, the antithrombotic responses varied among animals. In vitro supplementation of heparin fragments at 5 times the concentration which protected animals against the thrombogenic effects of activated prothrombin complex concentrates failed to produce any elevation of prothrombin time, partial thromboplastin times, thrombin time and other coagulant assays. Our studies suggest that ULMFs are potent antithrombotic agents and may exert their effects involving multiple sites and primarily inhibiting the Xa and the non-thrombin serine proteases formed during activated states.
Carrier Detection In Hemophilia I“A” Using Discriminant Analysis: A 5 Year Study
Authors: Graham J B , Barrow E S , Reisner H M
Published: July 2018

We counseled 174 possible or obligatory carriers of hemophilia “A” between January 1975-January 1980. Pedigree information provided a pedigree probability, πC, and blood samples the likelihood ratio favoring carriership (L.R.). The latter was arrived at from 2 bioassays of F. VIII activity (VIII:C & VIIIR:Ag) by linear discriminant analysis. Each consultand received counsel based on a final probability, P(C), produced by combining πC and L.R. Eighty % of the values of P(C) were either very high or very low which meant that clear-cut advice could usually be given. There were 29 obligatory carriers, 16 mothers of sporadic hemophiliacs, 73 sisters of hemophiliacs, and 56 more distant relatives. The individual values of P(C) could be used within each subset of carriers to relate mendelian expectation and observation. There were very few low P(C)s among mothers of sporadics suggesting that almost all were carriers. As a result, we now consider 0.8 to be a conservative πc for mothers of sporadics. Four women with low P(C)s were present among 29 obligatory carriers. This provided an estimate of negative diagnostic error (4/29=14%) and probably represented the effects of “lyonization”. The observedrexpected ratios of high:low P(C)s were normal among sisters of hemophiliacs and second and third degree relatives. Ten % of our consultands were pregnant when first seen. Twelve mothers with low P(C)s produced 11 liveborn, non-hemophilic children, 7 girls and 4 boys. Four with high P(C)s requested amniocentesis; 3 males were discovered and 2 were aborted. Five possible carriers with high P(C)s who had been seen before pregnancy, returned for assistance after becoming pregnant. All 5 requested amniocentesis, and fetoscopy if a male were present, and 4 males were fetoscoped. Two diagnosed as non-hemophilic in utero proved to be normal after delivery. A third could not be diagnosed, but was non-hemophilic at birth. The fourth, a hemophiliac, did not survive the procedure.
Evidence For A Second Molecular Defect In Hemophilia A
Authors: Brown J E , Hougie C
Published: July 2018

Immunoradiometric assays (IRMAs) for factor VIII coagulant antigen (VIII:CAg) utilize naturally occurring antibodies arising in hemophiliacs or previously normal individuals. Factor VIII:C loses its activity in the presence of these antibodies, and when they are used in an IRMA, little or no binding material, referred to as VIII:CAg, is detected in severe hemophilia A or von Willebrand’s disease plasmas. It is therefore generally assumed that such antibodies are specifically directed against a site on the VIII:C molecule. We have observed that factor VIII:C and VIII:CAg fractionate in different regions when gel-filtered factor VUI/vWf concentrates are chromatographed on QAE Sephadex; the vWf appears first, followed by VIII:CAg and then VIII:C. Sucrose density gradient ultracentrifugation on plasma from either a resting or exercised donor demonstrated two separate regions of activity with VIII:C sedimenting significantly faster than VIII:CAg. These separation experiments occurred using plasma prepared from blood collected into DFP and Trasylol and were obtained using two different antibodies in the IRMA. The VIII:CAg in a gel- filtered factor VUI/vWf concentrate was more heat labile (56°C) than VIII:C. These findings suggest that some of the antibodies used in the IRMA may not be directed against a site on the VIII:C molecule, but against sites on another protein (factor VIII coagulant related antigen?). A sequitur of this hypothesis is that there is a second protein deficiency in hemophilia A.
Immunoradiometric Assay Of VIII:CAG, A Potential Tool To Detect Human Anti-VIII:C Antibodies
Authors: Hellings J A , Over J , van Leeuwen F R , van Mourik J A
Published: July 2018

A modification of a two-site, solid-phase immunoradiometric assay (IRMA) for Factor VIII coagulant antigen (VIII: CAg) has been evaluated for its potential to detect antibodies against Factor VIII coagulant activity (VIII: C) in patient plasma samples. For this purpose the assay system comprised four steps: 1) coating of test tubes with human anti —VIII:C , 2) incubation with normal Factor VIII— VWF complex, 3) incubation with test sample, and 4) binding of radiolabeled human anti–VIII: C as marker.

Of eight hemophilic plasma samples containing antibodies against VIII: C (as detected in a clotting assay) five were able to prevent binding of radiolabel partially and three prevented this completely. One of these three (which actually was the antibody used in the IRMA), was effective even at very high dilutions. Two hemophilic plasma samples without detectable antibodies in the clotting assay, a severe Von Willebrand’s disease plasma and normal plasma samples showed no significant interference with binding of radiolabeled human anti-VIII: C. Also, a plasma sample containing a high titer of spontaneous human antibody to VIII: C as well as a heterologous antiserum against Factor VIII—VWF complex did not interfere with binding of radio-activity.

It is concluded that the test system described is a sensitive tool to detect antibodies of the same specificity as those used in the IRMA. It may also detect antibodies of differing specificity. The lack of crossreactivity with some antibodies points to interindividual differences in specificity of antibodies against VIII: C.
Polyelectrolyte Fractionated Porcine Factor VIII Concentrate In The Treatment Of Haemophiliacs With Antibodies To Factor VIII: C
Authors: Kernoff P B A , Thomas N D , Lilley P A , Tuddenham E G D
Published: July 2018

Antibodies to procoagulant factor VIII (anti-VIII:C) occurring in patients with haemophilia neutralise porcine factor VIII:C less readily than human factor VIII: C in vitro. Porcine factor VIII concentrate (porcine VIII) therefore has potential advantages in the treatment of such patients. Polyelectrolyte-fractionated porcine VIII (PE porcine VIII) lacks a major drawback of earlier preparations of porcine VIII in that it contains negligible quantities of platelet aggregating factor (PAF). The purpose of this study was to make a preliminary clinical assessment of the therapeutic value of PE porcine VIII. Over 6 months, 12 courses of treatment were given to four patients with circulating anti-VIII: C. Bleeding episodes treated ranged from the potentially lethal to moderately severe joint and muscle haemorrhages. Duration of courses was from 24 hrs. to more than 3 weeks. Clinical responses were strikingly good and no patient developed thrombocytopenia. Occasional mild pyrogenic-type transfusion reactions were encountered, but these were easily controlled. Dose- response relationships were most favourable in patients with low pre-infusion levels of anti-VIII: C activity against PE porcine VIII but excellent clinical responses could be obtained without achieving high plasma VIII: C levels. Multiple courses of therapy (up to 6) were given to individual patients without evidence of loss of clinical or laboratory efficacy, or an increased tendency to adverse reactions. There was no evidence of resistance in the patient who was treated daily for more than 3 weeks. Only 1 course of therapy was followed by a classical anamnestic rise in anti-VIII: C, and this course had included human factor VIII. PE porcine VIII appears to have a low immunogenic potential, and is a rational and effective therapeutic alternative for patients with anti-VIII: C.
Increased Circulating Immune Complex Levels In Hemophilia Patients Post-Infusion
Authors: Hilgartner M W , Inman R D , Miller C H
Published: July 2018

Elevated levels of circulating immune complexes (CIC) have been reported in a significant proportion of hemophilia patients tested by Raji cell, Clq binding, and staphylococcal binding (SBA) methods. The clinical significance of these complexes is unclear, as is their relationship to transfused material. By SBA, we found elevated CIC levels (>10 ug/ml) in 90% of severe hemophiliacs and 75% of multiply-transfused controls, all more than 72 hours post-transfusion. This was in contrast to our previous finding elevated CIC in 50% of hemophiliacs and 29% of controls by Raji cell assay.

Using the SBA, seven patients were tested at intervals after Factor VIII concentrate infusion. Five showed increased CIC levels. The peak level occurred after the Factor VIII clotting activity had peaked and was declining. Two subjects showed rapid clearance of the complexes, while the others remained elevated at 24 hours. Factor VIII-related antigen was detected in isolated complexes from 2 patients. Our previous finding of HBsAg in such complexes suggests heterogeneity and perhaps different clearance patterns.
Elimination Of Haemorrhagic Complications In Postoperative Thromboprophylaxis. Investigations On Heparin 2500 IU + Dhe 0.5 Mg
Authors: Buttermann G , Haluszcynski I , Theisinger W , Pabst H W
Published: July 2018

Dihydroergotamine (DHE) is known to enhance the effect of low-dose heparin in the prevention of postoperative thromboembolism. In order to determine whether low-dose heparin can be reduced without decreasing the prophylactic efficacy, 350 fully randomized patients undergoing major abdominal surgery were studied by the 1-125-fibrinogen-uptake-test (RFUT) and repeated lung perfusions scintigraphy receiving either 5000 IU of heparin or 2500 IU of heparin + 0.5 mg DHE b.i.d.

Results: Heparin 5000: 15 RFUT-positive (9.3%), 5 with lung perfusion defects (3.1%), 40 with haemorrhagic complications (24.8%); Heparin 2500 + DHE: 13 RFUT-positive (8.0%), 4 with lung perfusion defects (2.5%), 8 with haemorrhagic complications (4.9%), the difference in bleeding complications is highly significant (p < 0.001).

Conclusions: The combination of heparin 2500 IU + DHE 0.5 mg b.i.d. proved equivalent to heparin 5000 IU b.i.d. in terms of antithrombotic efficacy, whereas bleeding complications due to prophylaxis (wound haematoma, postoperative bloodloss, reoperations because of haemorrhage) were significantly reduced. Altogether this combination turns out an important progress in the postoperative thromboprophylaxis.
A Comparative Study Of Heparin And A Heparin Analogue In The Prevention Of Thrombosis In Patients With Gynaecological Cancer Under Radiotherapy
Authors: von Hugo R , Hafter R , Graeff H
Published: July 2018

The frequency of thrombosis (D.V.T.) in patients treated for gynaecological cancer by combined external irradation and intravaginal radium application, as detected by the 125 I-fibrinogen-test, is considerable (43 % D.V.T.).

84 patients undergoing radiotherapy were studied in a first controlled trial to assess the effect of 2×7500 U daily s.c. calcium heparin and 2×5000 U daily s.c. of a semisynthetic heparin analogue (SSHA) on the incidence of D.V.T. during therapy. 4 patients had to be excluded. The frequence of D.V.T. was 15 % (6/40) in the heparin group and 25 % (10/40) in the SSHA group. The difference was not statistically significant.

Long term application of prophylaxis (regular radiotherapy lasts 4 weeks) causes discomfort to the patient because of the numerous subcutaneous injections.lt was the aim of a second trial to investigate the prophylactic efficacy of a single s.c. injection per day.

80 patients were randomly allocated into two groups and received either 12500 U calcium heparin (n=40) or 12500 U SSHA (n=40). The incidence of D.V.T. was reduced to 15 % by a single heparin injection per day; in patients given SSHA the incidence was 17,5 %.

The results presented indicate that SSHA is as effective as heparin in preventing D.V.T. during radiotherapy and that a single s.c. injection per day is sufficient.
Use Of Indium-111 Labeled Platelets To Assess Platelet Deposition On Arteriosclerotic Plaques In A Primate Model
Authors: Welch M J , Mathias C J , Powers W J , Sherman L A , Siegel B A , Clarkson T B
Published: July 2018

Groups of primates (Macaca arctoides) were fed either a control (n=4) or a high-cholesterol diet (n=4) for a period of at least five years. Serum cholesterol levels in the experimental group were in excess of 250 mg/dl while the control group had levels from 150 to 200 mg/dl. In-111-labeled platelets and Tc-99m-labeled red blood cells were injected into each animal, and consecutive images were taken and stored in a computer immediately thereafter and 24 hours later. The Tc-99m-labeled red blood cell image was used to subtract the blood pool background from the In-lll-labeled platelet image to enhance visualization of platelet deposition. The subtracted images allowed the platelet deposition in the regions of the abdominal aorta, femoral, carotid, and renal arteries to be determined. All of the experimental monkeys hafi abnormal accumulation versus only one of the control animals. Four of the animal studies were repeated (2 experimental and 2 control), and comparable results were obtained. The animals were then given 3 mg/kg/day of aspirin for 5 days and the studies repeated. No abnormal accumulations of platelets were seen in the animals after treatment with low-dose aspirin (30 mg/kg/day) and prostacyclin (PGI2). Our results suggest that In-lll-labeled platelets may be useful to study the pathogenesis of arteriosclerosis and pharmacological techniques for its amelioration.
Locating And Monitoring The Activity Of Intracardiac Thrombi With Indium-111 Platelet Scintigraphy
Authors: Ezekowitz M D , Smith E O , Cox A C , Herren S W , Taylor F B
Published: July 2018

Indium-III is 2.8 day half-life gamma emitting radionuclide which is suitable for scintigraphic study and has been used to label platelets without causing significant attenuation of function. The purpose of this study was to utilize this technique for localization of left ventricular mural thrombi in patients with regional LV dysfunction. The patient population consisted of 55 patients between the ages of 24 and 77 (53.4 ± 11.3, mean ± 1SD). Twenty-four required coronary artery bypass surgery with aneurysmectomy for intractable angina and/or heart failure. This provided an opportunity to validate the preoperative findings at surgery. Platelets were separated from 43 ml blood in ACD solution by centrifugation and were labelled in ACD:saline (1:7) solution at a pH of 6.5-7.0. A total of 3.8 ± 2.9 × 109 (mean ± 1SD) platelets labelled with 451.9 ± 111.6 μCi with a final labelling efficiency of 72.1±14.1% were injected IV. Platelet recovery at 15 minutes was 51.1 ± 17.7% (mean ± 1SD). EM studies before and after labelling showed no morphological change due to the labelling procedure. Aggregation of platelets in response to ADP and collagen was not altered significantly during the labelling process. Patients on aspirin showed the expected inhibitory effect of aspirin on collagen and ADP induced aggregation. Patients were imaged in multiple views on at least alternate days for a maximum of 8 days. Seventeen had positive studies. In those patients in which surgical confirmation of the scintigraphic studies was possible, a sensitivity of 72% and specificity of 100% was found. We conclude that: 1) Indium-111 platelet scintigraphy promises to be a reliable method of identifying intracardiac thrombi. 2) It may also be useful in monitoring thrombus activity in vivo. 3) Patients on aspirin incorporated platelets onto the thrombus surface in spite of showing the expected inhibitory response to ADP and collagen induced aggregation in vitro.
The Effect Of Antiplatelet Drugs On Platelet Survival Time And Betathromboglobulin In Coronary Artery Disease
Authors: de Boer A C , Han P , Turpie A G G , Butt R , Gent M , Genton E
Published: July 2018

Platelets play a role in the development and complications of coronary artery disease (CAD). Abnormal platelet tests which can be corrected by antiplatelet drugs have been reported in CAD, including shortened platelet survival time (PST) and increased plasma betathromboglobulin (BTG) concentration. However, most of these reports were retrospective and unblinded. For these reasons we studied in a randomized double-blind crossover trial, the effect of sulphinpyrazone (800 mg) or aspirin (1200 mg)/dipyridamole (200 mg) on 51-Chromium PST calculated by an exponential model (normal 118±16 hrs; mean ± SD) and on plasma BTG measured by radioimmunoassay (28 ± 8 ng/ml) in 40 patients with angiographically documented CAD. Mean PST pretreatment (116 ± 16 hrs) was not different from normal; after 1 month’s treatment with placebo, sulphinpyrazone or aspirin/dipyridamole, mean PST was 123 ± 22, 123±17 and 128±26 respectively. Mean BTG pretreatment (52 ± 33) was elevated compared to normal (p<0.005) and abnormal in 21%; after treatment with placebo, sulphinpyrazone or aspirin/dipyridamole, mean BTG cone, was 59 ± 68, 51 ± 35 and 47 ± 33 respectively. Analysis of variance showed no significant differences between the various treatment groups for either PST or BTG. There was no correlation between PST and BTG (r= −0.04). In addition, there was no correlation of BTG or PST with the severity of CAD either clinically or angiographically. The data indicate that BTG levels were elevated in a proportion of patients with CAD and were not affected by the anti platelet drugs tested. The values of PST in CAD were usually within the range of normal, varied significantly between determinations and were not influenced by the anti platelet drugs.
Abnormal Arachidonic Acid Metabolism In Diabetes: Effect Of Normalisation With Drugs
Authors: Johnson M , Reece A H , Harrison H E
Published: July 2018

Patients with diabetes mellitus develop microvascular complications and have an increased susceptibility to both atherosclerosis and arterial thrombosis. We have demonstrated an imbalance in arachidonic acid metabolism in diabetes, vascular prostacyclin (PGI2) being reduced and platelet thromboxane (TxA2) being elevated. Platelet aggregation in response to ADP and arachidonic acid is also increased, and platelet survival is decreased. Treatment of diabetic rats with a specific thromboxane synthetase inhibitor, 1-nonyl-imidazole (50mg/Kg), for 7 days significantly (p<0.05) inhibited platelet aggregation and TxA2 synthesis. Aortic PGI2 production was increased and there was now no difference between treated animals (0.39 ± 0.05ng/mg) and non-diabetic controls (0.4l ± 0.04ng/mg). “Low dose” aspirin (2.5mg/Kg) further decreased PGI2 levels. Chronic treatment of diabetic animals for 3 months with 1-nonyl-imidazole restored both TxA2 and PGI2 production to normal, and significantly reduced the incidence and severity of microvascular lesions. If an imbalance in arachidonic acid metabolism plays a role in the vascular complications of diabetes, then the apparent beneficial effect of some drugs has important implications for therapy.
The Influence Of Cyclooxygenase Inhibitors On Peripheral Blood Flow In Man
Authors: Tooke J E , Tindall H
Published: July 2018

Previous work has shown that inhibition of cyclooxygenase with indomethacin and flurbiprofen blunts post-occlusive reactive hyperaemia in man. Patients with diabetes mellitus exhibit a similar control-related impairment in vascular response due to their underlying disease. We were therefore concerned that the use of cyclooxygenase inhibitors in these patients might further impair vascular reactivity. We studied nine insulin-dependent diabetics with retinopathy who were given either aspirin 330mg + dipyridamole 75mg tds or placebo according to a randomised double-blind cross-over protocol. Calf and digital perfusion were measured using an ECG-triggered mercury strain gauge pleth-ysmograph (Janssen, Peri flew) which provided a semi-continuous computerized calculation of blood flow. Rest flow (RF) was measured following full acclimatization in a constant temperature room. Peak flow (PF) was assessed from the reactive hyperaemia following a four minute period of arterial occlusion. The aspirin- dipyridamole combination caused a significant reduction in mean PF : RF ratio (9.2: 7.2, p<0.0l) measured in the calf, and a significant prolongation of the mean time for 50% decay of peak flow (l9.7 to 26.0 seconds, p<0.02). A significant reduction also occurred in digital PF : RF ratio following aspirin-dipyridamole (5-0: 3-7, p<0.05). These results suggest that moderate doses of cyclooxygenase inhibitors prescribed for their platelet-inhibiting action may have profound effects on vascular reactivity. In diabetic patients the effects may add to a pre-existent abnormality.
Endotoxin-Mediated Endothelial Injury In Vitro
Authors: Harlan J M , Harker L A , Striker G E , Counts R B
Published: July 2018

Endotoxin (ET) infusion into animals produces endothelial cell (EC) injury. It is not clear whether EC injury is produced by ET directly or by an ET-generated mediator, or whether ET-mediated EC injury also occurs in man and non-human primates. To answer these questions we examined the effects of ET on human umbilical vein (HUVEC) and bovine aortic (BAEC) endothelial cells in culture. ET (100 ug/ml) ± complement ± neutrophils produced no significant HUVEC Cr-release or cell detachment at 4 hours. HUVEC proliferation by direct cell count, angiotensin converting enzyme activity by labeled substrate conversion, VIII-Ag release by RIA and fibronectin release by ELISA were not affected by ET (100 ug/ml). ET alone did not induce significant release of PGI2 measured by bioassay or 6-keto-PGF. I α measured by RIA. In contrast to the lack of effect of ET on HUVEC, ET produced a time (3 hours: 22.0 ± 4.0%; 6 hours: 40.0 ± 6.0%; 24 hours: 71.0 ± 5.0%) and dose (100 pg/ml: 25.4 ± 2.5%; I ug/ml: 83.0± 4.0%; 10 ug/ml: 92.0 ± 3.0%) dependent BAEC detachment which was initially sublethal without 51 Cr-release or trypan blue dye uptake (40.6 ± 6.0% detachment, <1% Cr-release at 6 hours with ET 10 ug/ml). BAEC detachment was seen with all ET preparations including lipid A. ET-mediated BAEC detachment was inhibited by incubation at 4° but not by indomethacin, methyl prednisolone, or chi or promazine and was not dependent on serum or complement. Examination of ET-detached BAEC by SDS-PAGE after 125I- surface labeling or by immunofluorescence using anti-fibronectin antibody revealed complete loss of cell surface fibronectin. ET- mediated detachment was also seen with bovine mesenteric EC but not with bovine aortic smooth muscle cells or monkey aortic EC. These studies demonstrate the importance of studying EC from appropriate species and vascular sites. Although ET has no cytotoxic effect on HUVEC, it does induce selective, sublethal BAEC detachment associated with loss of cell surface fibronectin. If a similar effect occurs in human adult arterial or microvascular EC, it would have profound pathophysiologic effects accounting for the permeability changes and thrombosis seen in sepsis in man.
Vascular Smooth Muscle Cell Proliferation Caused By A Remote Injury
Authors: Stemerman M B , Gardner R T , Fuhro R
Published: July 2018

Proliferation of vascular smooth muscle cells (SMC) has been postulated to occur from blood-borne mitogenic stimuli. The hypothesis we tested states that SMC’s can be stimulated to enter DNA synthesis and proliferate by a humoral material (s) released from a remote site. Rabbits aortas were bat loon de-endothelialized in the following manner: Animals were ballooned throughout their aortas at day 0 except Group IV; Group I were not re-injured thereafter, Group II, only the abdominal aortas were re-ballooned at day 4, Group III were sham-operated (femoral artery tied-off) day 4, and Group IV underwent de-endothelialization of only the abdominal aortas on day 0 and 4. Animals were sacrificed by ex-sanguination on days 4, 4.5, 4.75, 5, 6, 7, 14 and 28. One hour prior to sacrifice, each animal received 0.5 uCi/gm of 3H-Thymidine I.V. and 1/2 hr later 5 ml of Evans Blue dye. Within one minute following death, the thoracic segment (3rd to 6th intercostal arteries) was removed and processed for SMC DNA specific activity (SA). Adjacent throacic segments as well as abdominal aortas were immersed in 2.5% gluteral- dehyde and processed for morphology and morphometry. Group II SMC SA showed a sharp rise beginning at day 4.5 and peaked at day 5 (335 ± 62) returning to control levels by day 7. Comparison of Group II with Groups I and III respectively at day 5 (86 ± 19, 42 ± 8) were significantly different p< .01. Group IV animals showed no rise in SA. Intimal cell nuclei per 0.1 mm internal elastic lamina, counted at day 7, showed significant differences also (p <.01); Group II 9 ± 0.7 Group I 5 ± 0.3. These data demonstrate that rabbit aortic SMC’s can be caused to proliferate by a remote vascular injury likely due to a humoral substance(s) perhaps released from platelets. Thus, direct vascular injury with release of potential mitogens into the vessel wall underlying the injured tissue is not a prerequisite of SMC proliferation and myointimal growth.
Inhibition Of Platelet Aggregation And Release Reaction By A Calmodulin Interacting Agent, N-(6-Aminohexyl)-5-Chloro-1-Naphthalene Sulfonamide
Authors: Suda J , Aoki N
Published: July 2018

Platelet myosin light chain kinase is a calmodulin dependent enzyme which requires calmodulin for its activity and is intimately related to actomyosin contracting system. Platelet actomyosin contraction may cause the release reaction of platelet. N-(6-aminohexyl)-5-chloro-naphthalene sulfonamide (W-7) is one of the calmodulin interacting agents, and selectively inhibits calmodulin activity. Since platelet membrane phospholipases which are responsible for the release of arachidonic acid from membrane phospholipids may also be calmodulin-dependent enzymes, the inhibitory effects of W-7 on platelet function were examined. W-7 in a concentration as low as 10 7#x03BC;M inhibited platelet aggregation and release reaction induced by ADP, collagen, epinephrine, thrombin, arachidonic acid and Ca2+-ionophore A23187. When platelets were preincubated with W-7 before addition of an aggregating agent, the inhibition became more pronounced. In ADP-induced aggregation, only the second phase was inhibited. Arachidonate metabolites (PGG2/H2 and thromboxane A2) failed to induce aggregation and release reaction of platelets preincubated with W-7. Furthermore, release of arachidonic acid from platelet membrane phospholipids induced by A23187 in the presence of indomethacine was inhibited by W-7. W-7 had no effect on glucose metabolism in platelets. The c-AMP levels in platelets were not significantly changed by incubation with W-7. These results suggest that W-7 inhibits directly phospholipase A2 and/or phospholipase C in addition to myosin light chain kinase. These results may in turn indicate that calmodulin is involved in platelet aggregation and release reaction mechanism including membrane phospholipases and the target system of thromboxane A2 (possibly actomyosin contracting system).
Studies On Calcium-Activated Neutral Protease In Platelet
Authors: Sakon M , Ohno H , Kurokawa E , Kambayashi J , Kōsaki G
Published: July 2018

It has been reported that platelet aggregation or release of arachidonic. acid in stimulated platelets is inhibited by protease inhibitors or by synthetic substrates for proteases, suggesting possible involvement of unknown proteases in the platelet reaction. The present study was initiated to identify such proteases in platelets.

Subcellular fractions of bovine platelets were obtained according to the method of Barber and Jamieson and caseinolytic activity was studied in each fraction at pH 8.0 in the presence or absence of Ca2+, using fluorescamine method. A significant caseinolytic activity was detected in the soluble fraction but none in the fractions of plasma membrane or pellet. Then, this protease was partially purified from the soluble fraction by means of ammonium sulfate fractionation and subsequent column chromatographies on DEAE-Sepharose CL-6B and Sephadex G-150. The partially purified protease required Ca2+ strictly for its activity with the optimal concentration of 1 mM and pH optimum for the activity was 8.0, by which this enzyme was classified as a calcium-activated neutral protease. The molecular weight was estimated to be 135,000 by Andrew’s method. The activity of the protease was strongly inhibited by sulfhydryl reagents but no enhancement of the activity was observed in the presence of cysteine or reduced glutathione. The inhibition of the activity by PMSF or DFP was observed only when the protease was preincubated at 25°C for 1 hour in the presence of Ca2+.

From these observations, it may be suggested that the enzyme may possess calcium binding site independent of the catalytic site and that by changing the conformation of the enzyme Ca2+ may convert the enzyme from an inactive form to an active form. However, the physiological role of the enzyme in the platelet reaction has yet to be fully elucidated.
Platelet Shape Change And Cytoskeletal Reorientation During Adhesion And Spreading
Authors: Mattson J C
Published: July 2018

Sequential morphologic and cytoskeletal changes which occur during platelet adhesion have been examined by transmission electron microscopy of detergent extracted whole mounts. Human platelets allowed to spread on formvar-coated grids for 5, 15, 30 or 60 min. were fixed in glut- araldehyde to which the non-ionic detergent Nonedet P40 had been added. Whole mounts examined after 5 min. of contact demonstrated “dendritic platelets” with centralized organelles and numerous elongated pseudopodia. The cytoskeletal organization was oriented around the pseudopodia with bundles of 60-120 Å filaments extending into each pseudopod. At the base of many pseudopodia dense filamentous mats measuring from 0.2-0.5 μ were intimately associated with the filament bundles; filaments appeared to radiate from filament mats producing stellate configurations. An underlying trabecular network of 30-85 Å filaments was present interconnecting the longer filaments. The microtubular coil usually associated with the centralized granulomere of aggregated platelets appeared to uncoil in adherent platelets. As adhesion progressed, platelets were seen to spread on the formvar surface. Web-like extensions of platelet cytoplasm filled in and connected pseudopodia eventually replacing these processes with large veils of extended cytoplasm. By 60 min. most platelets were completely spread and had circular, fanshaped or polygonal configurations. Radially oriented filament bundles extending from dense filament mats remained visible in the cytoplasmic veils of early spreading platelets, but as spreading progressed reorganization of filaments and filament mats occurred. Filament mats, in more completely spread platelets, were present at the platelet margin and filament bundles were no longer radially oriented but assumed a circumferential organization surrounding the centralized platelet granules. These studies demonstrate that cytoskeletal reorganization is intimately associated with the platelet shape change.
Release Of Platelet Factor-4 In Vivo After Heparin Injection
Authors: Rao A Koneti , Holt John C , James Pranee , Niewiarowski Stefan
Published: July 2018

A single bolus of heparin administered to 8 normal volunteers resulted in a significant increase in levels in platelet poor plasma (PPP) of platelet factor-4 (PF4) but not low-affinity platelet factor-4/β-thromboglobulin (LA-PF4/βTG). However, the presence of heparin interfered with the binding of 125I-PF4 to antibody in radioimmunoassay (RIA). This effect was overcome by increasing the concentration of NaCl from 0.15 to 0.5 M in the buffer used for RIA. In order to establish that the increased amount of immunoreactive material present in PPP was indeed PF4, the protein was isolated from postheparin plasma. A bolus of 5000 units of porcine lung heparin (Upjohn) was administered intravenously to 2 volunteers and plasma samples obtained before and 5 minutes after the injection. The levels of PF4 in PPP rose from 18 and 10 ng/ml before to 185 and 454 ng/ml at 5 minutes after injection in the two volunteers, respectively. The 5 minute samples were adsorbed to heparin agarose columns and PF4 levels decreased to 16 and 10 ng/ml respectively. The immunoreactive material was eluted with 1.2 M NaCl from the heparin agarose columns, showing typical elution pattern for PF4. This material was applied to SDS-polyacrylamide gel electrophoresis in parallel with purified PF4 obtained from human platelets. RIA carried out on eluates from gel slices revealed a species of the same molecular weight as standard PF4. Thus, heparin injection results in appearance in the circulation of a material identical to PF4. LA-PF4/βTG and PF4 are located in same granules and released in parallel during platelet stimulation. Further, LA-PF4 is cleared from plasma 4 times slower than PF4. Therefore, the elevation of PF4alone suggests release from sites other than platelets.
Relationship Between Arachidonic Acid Content Of Blood Platelets And Their TXA2-Propduction Upon Stimulation
Authors: Hornstra G , Haddeman E , Don J A
Published: July 2018

There is good evidence that the potency of activated platelets to produce thromboxane A2 (TxA2) is of primary importance in the regulation of arterial thrombus formation. Therefore it is useful to know the factors that determine platelet TxA2-production. In order to investigate the importance of the platelet arachidonic acid (AA) content in this respect, 6 groups of 24 rats were fed various diets which were likely to induce different platelet AA-contents. After 15 weeks of feeding, platelet rich plasma (PRP) of 4 rats were pooled. In one part of each PRP pool aggregation was triggered with four doses of collagen. After 5 minutes the aggregated PRP was processed for measuring the HHT, produced by the activated platelets, (CG/MS) which is considered a measure for the TxA2 produced. The other part of the PRP was used for measuring the fatty acid composition (GLC) of the 5 major phospholipid (PL) classes (separated by two-dimensional TLC). The various diets did not cause major changes in platelet total PL content and PL class distribution. Highly significant changes in the fatty acid compositions of platelet PL classes were observed. A striking positive correlation was found between the lyso-lecithin and the AA-con- tent of blood platelets, which suggests a functional interaction between these two parameters. There was also a highly significant, positive correlation between the AA-content of platelet PL and the TxA2-production by these platelets. Platelets of sunflowerseed oil fed animals did not fit this relationship because their TxA2-production is too high in relation to their AA-content. The relationships are essentially similar for total PL and the various PL classes and do not change when taking into account the presence of other long chain polyunsaturated fatty acids, known to compete with AA for the cyclo oxygenase enzyme system. From this study it is apparent that the platelet AA-content is an important determinant of TxA2 formation.
Further Studies On The Purified Phospholipase A2 From Human Platelet Plasma Membranes
Authors: Apitz-Castro R , Cruz M R , Mas M , Jain M K
Published: July 2018

The activation of a phospholipase A2 has been postulated in the release of arachidonic acid from phospholipids of platelet plasma membrane. The present communication deals with the biochemical properties of a phospholipase A2 isolated from human blood platelets. Phospholipase A2 from human platelets have been isolated to homogeneity through an acid or salt extraction, followed by affinity chromatography purification. The enzyme is copurified with the platelet plasma membrane, It has a MW of about 50,000 Dalton. It shows an optimum pH at 9.4 and requires calcium for its activity. Although it attacks a variety of phospholipid substrates, there is a preference for unsaturated phospholipids. Diarachidonoy1-PC liposomes can be hydrolyzed in the absence of any additive. The specific activity is markedly affected by the quality of the interface, showing variations of more than 10 fold between different substrate forms. The purified enzyme is considerably more active on the aggregated form of the substrate than on the monomeric form. Saturation behavior, consistent with Michaelis-Menten type of kinetics is observed at higher concentration of the micellar substrate. A four-fold increase in rate is observed when the enzyme is assayed in the presence of fatty acid + lysophospholipid. Maximal rates are obtained at a 20 mole percent of products to substrate. 1,2-diglyceride and phosphatidic acid stimulates the hydrolysis of phosphatidylcholine by the purified enzyme, however, in these forms of substrate, neither the diglyceride nor phosphatidic acid is hydrolyzed. Under optimal condition, the phospholipase A2 activity corresponds to at least 13 nmol/min/109 platelets. Activation of this enzyme by some intermediate related to the phospholipase C pathway might play a role in the stimulus- linked release of platelet arachidonic acid.