bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient
Authors: Rendu F , Lebret M , Caen J P
Published: July 2018

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.

Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.

The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.
Albumin And Platelets
Authors: Forestier F , Cherbit G , Solé Y , Tersarkissian M H
Published: July 2018

We set up to point a double antibody radio-immuno-assay to quantify platelet albumin content.

We used commercial reagents such as an antibody of rabbit reacting against human albumin and a highly purified human albumin antigen, labelled with 125I./Chloramine.

The main results were:
- 109 platelets contained approximately 1 mg of albumin

Zonal ultra-centrifugation (105.000 g) in saccharose gradients showed that albumin was located in non-lysosomal α granules.

The release of albumin occurred at the same time as β Thrombo-Globulin and Platelet Factor 4.

We studied the serum albumin (SA), platelet albumin (PA) and the number of platelets on 144 normal individuals aged from 20 to 90 years.

Our data show that:
- PA decreases in correlation with the platelet count

the ratio PA/SA increases with age

a correlation between the level of SA and the platelet counts exists.

These data suggested a regulation mechanism between serum albumin and platelet levels. On the other hand our findings proved an active transport of albumin through the platelet membrane.
Energy Control During Platelet Secretion:Predominant Role Of ATP-Turnover
Authors: Deijns A , Akkerman J W N
Published: July 2018

Platelet aggregation and secretion of granular contents require metabolic energy. This implies the existence of a control mechanism that adjusts the rate of energy producing pathways to the energy need of these functions. Such a control function has been attributed to the level of metabolic ATP, to the adenylate energy charge (AEC =(ATP + 1/2ADP)/(ATP + ADP + AMP) and to the velocity of energy generation and consumption (ATP-turnover). In this study we investigated which factor dominates in control of dense granule (3H-serotonin)-, α-granule (β thromboglobulin)- and lysosomal granule (N acetyl β glucosaminidase) secretion. Human gel- filtered platelets were incubated in glucose-free, CN containing medium. Under these conditions ATP-generation only took place in glycogenolysis, which alone was unable to maintain ATP homeostasis. Consequently, metabolic ATP and AEC fell. Addition of glucose restored glycolytic ATP resynthesis wich restored the AEC but the loss of ATP was for the main part irreversible due to hypoxanthin formation. With this system a broad range of metabolic ATP levels and AEC’s could be obtained both at decreasing ATP turnover (before glucose addition) and at increasing ATP turnover (after glucose addition). Analysis of secretion velocity of dense-, α- and lysosomal granules after initiation with thrombin (5 U/ml) showed that at decreasing turnover the secretion velocity of all types of granules depended on both metabolic ATP level and AEC. In contrast, at increasing ATP turnover the correlation between secretion velocity and metabolic ATP level or AEC was lost. Instead, dense granule secretion was faster and lysosomal granule secretion was slower than expected on the basis of metabolic ATP level or AEC, whereas α-granule secretion showed intermediate levels. The data indicate that the three types of granule secretion differ in their dependence on metabolic energy and that apart from metabolic ATP and AEC, ATP turnover is of crucial importance for secretion of granular contents.
Human Blood Platelets Cause Collagen Gel Retraction
Authors: Kopeć M , jeleńska M
Published: July 2018

Contraction of collagen gels under the influence of human blood platelets was studied and compared with platelet-mediated fibrin clot retraction. Soluble type 1 collagenfrom the rabbit skin was allowed to polymerize in the presence of washed platelet suspensions. Collagen gels formed in the presence of intact platelets retract with concomitant exclusion of the fluid. Collagen gel retraction is positively related to platelet numbers/5- 60×10 platelets/ml/ and negatively to collagen concent- rations/0.5 to2.0 mg/ml/. Under optimal conditions collagen gels shrink to about 12% while fibrin clots-to 8% of initial volumes. Similarly to fibrin retraction collagen shrinkage is abolished by disruption of platelets or chelation of Ca++ ions .Collagen retraction is inhibited by cytochalasin B /5μg/ml/ and N-ethylmaleimide/25μg/ml/. Colchicine,indomethacin, PGE1 , dipyridamol and serpa- sil did not influence collagen gel retraction. Thrombin Defibrase, Arvin and hirudin remain also without effect. Platelets from patients with severe deficiensies of f. VIII or of vWillebrand factor exhibit normal collagen retracting potency. Platelets from patients with Glanzmann’s thrombasthenia,defective in fibrin retracting capacity, provoke collagen shrinkage of nearly the same degree as platelets from healthy people .Certain tumor cell lines inactive in fibrin retraction have the potency to contract collagen. The presented data can be relevant to wound healing and tumor progression.
The Effect Of Metal Ions On Human Factor IX Antigenicity
Authors: Lewis R M , Reisner H M , Abels B C , Roberts H R
Published: July 2018

Affinity chromatography of an inhibitor to human factor IX (F.IX) separated the antibody into two populations. The ion dependent population of antibodies had an absolute divalent cation (Me++) binding requirement. The non-ion dependent population bound F.IX equally in the presence or absence of Me++. The concentration of Me++ required for ½ the maximum ion dependent antibody binding (½ max) was (in nM) Ca++ 0.40, Mn++ 0.05, Sr++ 0.70 and Mg++ 0.65.

Ca++ potentiated the binding of antibody in the presence of excess Mg++. In addition, the ½ max for Ca++ was reduced about four fold. These observations are consistent with separate binding sites on the F.IX molecule for Ca++ and Mg++ and potentiation of Ca++ binding by Mg++. Scat- chard analysis of ion dependent antibody binding indicates about a 10 fold greater affinity of antibody in the presence of Ca++ than Mg++. In the presence of both cations, affinity was at least as high as in the presence of Ca++ alone supporting the presence of separate ion binding sites on the F.IX molecule.
A typical Response To Infusion Prior To The In Vitro Appearance Of Inhibitor In A Hemophilia B Patient
Authors: Miller C H , Hilgartner M W
Published: July 2018

Infusion studies were performed on six patients with severe hemophilia B, in whom factor IX clotting activity (IX: C) was less than 1%. All had no detectable factor IX antigen (IX: Ag) on testing with heterologous anti-IX prior to the study (and no measurable F. IX inhibitor). Each patient received the same factor IX concentrate in a 50 u/kg dose. Samples were drawn at 0, 15 minutes, 1 hour, 4 hours, and 24 hours post-infusion and were tested for IX: C and IX: Ag. Five of the patients showed similar response to transfusion with average half-life of IX: C of 12 hours and of IX:Ag 10 hours. One patient showed quite different results. He had a half-life of IX: C of 31 hours and of IX:Ag of 24 hours. Although routine tests for a F. IX inhibitor were negative both before and after the study, he developed a rapidly rising inhibitor 15 months later. In retrospect it can be seen that this patient resembled the patient with a low titer inhibitor and prolonged IX: Ag survival described by Goodnight et al. However, our patient showed the atypical infusion response pattern before his inhibitor was detectable in vitro. Thus the finding of a prolonged survival of IX: Ag can be taken as evidence of inhibitor formation. Inhibitor presence can be confirmed by detection of circulating immune complexes containing IX: Ag prior to the appearance of a measurable inhibitor to clotting.
In Vivo And In Vitro Comparisons Of Anti-Inhibitor Coagulant Concentrates (AICC) For Hemophilic Therapy
Authors: Pancham N , Fournel M A , Coan M H
Published: July 2018

In FVIII:C deficient persons, who also have circulating antibodies to the molecule, replacement therapy with FVIII concentrates elevates the antibody level and is of limited or no value. Treatment with prothrombin complex concentrates, however, has had varying degrees of success in these patients depending on the significance of the bleed.

Currently available Anti-Inhibitor Coagulant Concentrates (AICC), containing factors II, VII, IX, and X in their nonactivated as well as activated forms, have been shown to be effective in controlling bleeds in some of these patients.

Two commercially available AICC products and two research preparations were evaluated using conventional in vitro clotting techniques. In vivo assays were also performed in animal systems using the Wessler venous stasis assay. Despite similarities by in vitro assays, striking differences exist in the results obtained using the animal systems. As shown by the Wessler venous stasis assay for in vivo testing, the materials are much more active (at least an order of magnitude) than currently manufactured factor IX concentrates.

In vitro clotting assays that are now available (namely factors II, VII, IX, X, IIa, Xa, NAPTT, FVIII correctional activity, and FVIII inhibitor bypassing activity assays) do not necessarily predict the in vivo efficacy of these AICC products. Furthermore, there is no correlation between the FVIII correctional activity and the FVIII inhibitor bypassing activity assays which are now being used to monitor these preparations.
Monoclonal IGM Anticoagulant With Acquired Abnormalities Of Factor VIII
Authors: Andes W A , Stuckey W J
Published: July 2018

An IgM tumor marker was studied for its effects on coagulation and factor VIII related antigen (FVIII R: Ag). A 62 year old man was found to have a lymphocytic lymphoma. Abnormal coagulation studies (Table) and a monoclonal gammopathy were noted but no abnormal bleeding. One course of chemotherapy induced a clinical response, normalization of coagulation studies and a rapid fall in IgM from 1110 to <30 mg% within 34 days. IgG and IgA were low. Additional chemotherapy resulted in further clinical improvement and coagulation studies remain normal (>11 mo). Mixing studies with plasma or purified IgM revealed an immediate acting, circulating anticoagulant against factors VIII and IX. No effects on thrombin, factors V, VII, X, or ristocetin cofactor activity could be demonstrated. FVIII R: Ag was increased to 220%. FVIII R: Ag was initially grossly abnormal on crossed immunoelectrophoresis. Both a normal and rapidly migrating component were seen as distinct peaks with approximately equal concentration. The faster component disappeared after chemotherapy. These studies suggest that (1) abnormal proteins may influence factor VIII functions and should be considered in the diagnosis or management of von Willebrand’s disease or acquired factor VIII inhibitors; (2) circulating anticoagulants may unpredictably influence coagulation proteins and may be tools in the investigation of hemostatic mechanisms.
Platelet Activation During Exercise Induced Myocardial Ischaemia
Authors: Hughes Andrew , IIsley Charles , Wilkinson Linda
Published: July 2018

Twenty six normal subjects (aged 18-52) and eighteen patients with ischaemic heart disease (aged 38-67) underwent maximum 12 lead ECG treadmill exercise testing. Coronary angiography was performed on sixteen of the patients. In vivo platelet activation was assessed by measuring plasma levels of the platelet-specific released protein beta-thromboglobulin (B-tg).

The mean B-tg levels pre and post exercise in the normal subjects were 47 (SD:26-85) ng/ml and 48 (29-78) ng/ml respectively, and in the patient group 68 (43-107) ng/ml and 70 (45-109) ng/ml. There was a significant difference between the two groups (p < 0.025), but there was no change in either following exercise. The mean B-tg level in those patients with positive exercise stress tests (≤0.1 mV ST depression at 80 ms post J point) was 75 (52-109) ng/ml, but only 46 (26-82) ng/ml in those with negative tests (p < 0.05). B-tg levels showed no change with increasing exercise ischaemia as judged by ST segment depression, but there was a positive correlation with the extent of angiographically demonstrated coronary disease (r = 0.497, p < 0.05).

We conclude that in vivo platelet activation is present at rest in patients with coronary artery disease, and whilst related to the presence and extent of such disease is unrelated to induced ischaemia.
Effect Of Gradual Coronary Artery Occlusion On Platelet Function In A Dog Model
Authors: Chesney C M , Scheel K W , Wilson J
Published: July 2018

The effect of gradual coronary artery occlusion on in vitro platelet aggregation as well as the “in vitro” platelet aggregate ratio of Wu and Hoak and was studied serially in a dog model. Healthy male adult mongrel dogs were subjected to thoracotomy. The circumflex artery was isolated and an Ameroid constrictor applied. This device consists of hygroscopic material which swells and occludes the artery gradually over a 14 day period (Circ. Res.40:384 1977.)Ten dogs were followed for 90 days. Platelet aggregation with ADP (10-3,10-5 and 10-6M) as well as the platelet aggregate ratio (normal=0.8-1.0) were determined preoperatively and postoperatively at 7,14,30 and 90 days,respectively. After blood was obtained on day 90 the dog was anesthetized. The beating heart was removed and transferred to an isolated heart perfusion system. The 3 coronary vessels were cannulated and flow to each vessel was measured simultaneously under constant pressure perfusion. Collateral flow was assessed by the retrograde flow method. At the termination of the study hearts were perfused with buffered formaldehyde-gluteraldehyde. Cross-sectional slices were obtained and stained with Mallory stain. The infarct size was measured relative to the left ventricle size. There was a strong correlation (r= -0.8683) between the infarct size and platelet aggregate ratio at day 14, the time of total occlusion. This data relates decreased aggregate ratio (reflecting an increase in circulating platelet aggregates) to increased infarct size. There was also a correlation between platelet aggregation(10-3M ADP) preoperatively and at day 14 (r = 0.7508). The sensitivity of platelets to ADP (10-5M) increased significantly at 90 days (p<0.0004). This increase in sensitivity did not correlate with infarct size or collateral flow, suggesting a different mechanism of platelet activation.
Clot Impedence As An Indicator Of Platelet Dysfunction Following Cardiopulmonary Bypass
Authors: Saleem A , Yawn D H , Saleh S A , Crawford E S
Published: July 2018

Post-operative bleeding following cardiopulmonary bypass remains a serious problem. Recent studies have indicated platelet dysfunction may be responsible for altered hemostasis in a significant number of patients. Although evaluation of coagulation factors can usually be done with speed and precision, evaluation of platelet function is time-consuming. We have evaluated a clot impedence device (Sonoclot®, Sieneo Inc., Colorado) to measure platelet function. The device measures and records the clot impedence to a vibrating probe as the blood sample clots and retracts. In our evaluation of healthy subjects, we found the initial slope of the impedence curve and the entire retraction phase are influenced by the number of platelets. Extrapolating this information to the patients undergoing cardiovascular bypass, we found 7 out of 11 patients with postoperative bleeding had poor retraction phase in spite of an adequate platelet count. This suggested platelet dysfunction. All seven patients achieved satisfactory hemostasis after platelet transfusion. This was correlated with a normal clot impedence study. Four patients with normal clot impedence were found to have surgical bleeding. The test is easy to perform and the result is available within fifteen minutes of drawing the blood sample. In our hands, the measurement of clot impedence appears to be a reliable adjunct in the etiological diagnosis of post-operative bleeding.
Hyperaggregability Of Serotonin-Depleted Platelets To Arachidonic Acid (AA) In SLE And Transplant Recipients
Authors: Marchesi D , Parbtani A , Frampton G , Livio M , Remuzzi G , Cameron J S
Published: July 2018

In a variety of renal disorders, including SLE and transplantation, in vivo platelet activation is present. This is in part a result of the nephrotic syndrome, but is also observed in patients with g.n. who are not nephrotic. As a result, intraplatelet concentrations of serotonin and other amines are depleted in patients with active nephritis. The functional capacity of such amine-depleted platelets has been little studied; Pareti et al (1980) reported defective aggregation to ADP, adrenaline and collagen in such “acquired storage pool deficiencies”. We studied aggregation thresholds to ADP, collagen and AA in 25 patients with SLE nephritis, none of whom had a nephrotic syndrome or reduced renal function. In these patients platelet activation is due principally to circulating factors. Sixteen transplant recipients were also studied. In them, activation is principally within the renal microvasculature of the allograft. We also measured intraplatelet serotonin concentrations, which were low or zero in the great majority of patients in both groups. Bleeding times were normal in all, as were platelet aggregation thresholds to ADP and collagen. AA thresholds, however, were markedly depressed (<0.32 mmol) in all transplant recipients and most patients with SLE. This specific hyperaggregability may be responsible for both persistence of disease and the thrombotic tendency observed in some patients, including those with circulating anticoagulant.
Thrombin Binding Activity Of Intact Platelets And Tumor Cells
Authors: Tullis J L , Hewitt L
Published: July 2018

Screening studies on the amount of thrombin bound to intact human platelets show that deficiencies are present in certain disorders which are associated with hypercoagulable states. Among the hematologic diseases where this is found most prominantly are primary thrombocythemia and polycythemia vera. A direct relationship can be plotted between the decrease in thrombin binding and the proportion of abnormal platelets present in the circulation. A deficiency also is present as an acquired disorder in occasional patients with advanced cancer in whom spontaneous thrombotic events complicate the clinical course. These include adenocarcinomas of the breast, colon and lung. It does not appear to be present in patients with epidermoid cancers. Whether these platelet changes bear a cause and effect relationship between growth and dissemination of the specific neoplasms or are merely a consequence of a generalized hypercoagulable state is unknown. However, the conditions in which the thrombin binding potential of the platelets is decreased coincide closely with separately published findings in which the thrombin binding potential of adenocarcinoma cells was shown to be different from epidermoid carcinoma and comparable normal control tissues.
Inhibition Of Platelet Release Reaction By Cytidine Monophosphate (CMP): Correlation With Inhibition Of Membrane Sialyltransferase Activity
Authors: Wu K K , Ku C S L
Published: July 2018

To provide further evidence for the contention that platelet surface sialyltransferase plays a major role in platelet release reaction, we evaluated the effect of CMP, a sialyltransferase inhibitor, on platelet aggregation and release. CMP exerted a dose-related inhibitory effect (IC5050=1.5mM) on the basal enzyme activity of intact washed platelets or membrane preparation. It blocks the enzyme stimulation by collagen, thrombin, ADP and U46619, a thromboxane A2 agonist. Serial experiments were then carried out in a lumiaggregometer to determine its effect on platelet release and aggregation. Pretreatment of platelet rich plasma or washed platelet suspension with CMP resulted in a significant reduction of ATP release induced by thrombin, collagen, ADP, U46619 and arachidonate. The dose-response curve of release inhibition was parallel to the enzyme inhibition. Platelet aggregation induced by ADP, collagen and sodium arachidonate was significantly inhibited by CMP but the dose-response curve shifted to the right. Platelet aggregation induced by thrombin or U46619 was unaffected. To determine whether the inhibitory effect of CMP might be mediated through stimulation of adenylate cyclase, platelet cyclic AMP content was determined by radioimmunoassay. There was no significant difference in the cyclic AMP content between CMP-treated and control platelets. CMP did not cause liberation of lactic dehydrogenase from platelets. These findings clearly indicate that inhibition of release reaction by CMP is linked to the inhibition of surface sialyltransferase and is independent of cyclic AMP. This study confirms the notion that platelet membrane sialyltransferase is involved in the initiation of platelet release reaction.
The Effect Of PF-4 Peptide Analogues On Platelet Activation And Release
Authors: Walz Daniel A , Fareed Jawed , Bajusz Sandor
Published: July 2018

The complete amino acid sequence of human PF-4 has demonstrated it to be a 70 residue protein with a lysine-rich carboxy terminal region. Chemical data, drawn from specific modification of these lysine residues, has been strongly suggestive of the dominance of these lysines in the interaction of PF-4 with heparin. We have synthesized argiHyland lysyl-peptide analogues of the human PF-4 sequence, residues 58-68, to mimic the putative heparin binding domain. Peptides 1 and 5 are the most effective samples in plasma for heparin neutralization. When each peptide was incubated with platelet rich plasma (PRP) in submicromolar concentrations, and followed by the addition of ADP, peptides 1-4 but not peptide 5 quantitatively blocked the release of PF-4 as judged by radioimmunoassays of the PRP supernatant. Peptide 1 also supressed the second aggregation wave follow ing ADP stimulation. Upon completion of aggregation of PRP, exogenously added PF-4 or these peptide analogues appeared to promote clot retraction. In other experiments, PF-4 and its peptide analogues induced a strong contraction in isolated uterus and ileum preparations. We are presently evaluating the ability of these analogues to block the interaction of PF-4 with its platelet proteoglycan carrier. This may mean that PF-4 or its analogues interact directly with cellular contractile elements and thereby modulate their contractile response.
Immunology Of The Platelet Surface
Authors: Rosse W F , Mendelson D S , Guibout C
Published: July 2018

Platelet-bound IgG, measured by quantitative anti globulin consumption, may be due to: 1. Heteroimmune reactions - the antibody binds to foreign antigen which binds to platelet surface either specifically (quinidine) or (possibly) not. 2. Alloimmune reactions - platelet- specific antigens (PIA1) are located on glycoproteins IIb- III; HLA antigens are located on two-peptide units similar but not identical to HLA units on lymphocytes. 3. Autoimmune reactions - the antigens have not been identified chemically or serologically but some are present on glycoproteins Ilb-III, others are on other (minor) proteins. 4. Immune complexes - these are presumably bound through the Fc receptor of platelets, the chemical identity of which is not known. 5. “Non-immune” adherence IgG is normally bound to four proteins of the platelet surface, including the major glycoproteins Ilb-III. The binding of supranormal amounts of IgG to normal platelets initiates the release reaction if sufficient is bound; this occurs regardless of which type of reaction outlined above leads to its binding. This results in the release of PF3 and serotonin and the aggregation of the platelets. Complement (C) is bound by some but not all IgG antibodies; immune complexes and fluid-phase activation may also bind it. The site of C3 binding is not known. The fixation of C3 can cause a release reaction similar to that seen with IgG binding. Sufficient bound C3 to mediate this is difficult to attain with normal platelets. Since the platelets of patients with paroxysmal nocturnal hemoglobinuria fix much more C3 for a given level of complement activation, they are much more likely to undergo these reactions than normal cells. This may account for the thrombotic tendency in these patients. Complement may lyse platelets by insertion of the terminal complex through the membrane surface; this is more easily accomplished on PNH than normal platelets.
Hemodynamic Shear Stress And Endothelial Cell Function: In Vitro Studies
Authors: Gimbrone M A , Dewey C F , Davies P F , Bussolari S R
Published: July 2018

The vascular endothelial lining in vivo is constantly subjected to hemodynamic shear stresses resulting from normal and altered patterns of blood flow. To facilitate the study of effects of fluid shear stress on endothelial cell structure and function, we have developed an in vitro system, utilizing a cone-plate apparatus, to subject coverslip cultures of bovine aortic endothelial cells (BAEC) to controlled levels of shear (up to 102 dynes/cm2) in either laminar or turbulent flow. The magnitude and direction of shear stress within the system are accurately known from both theory and experimental measurements. The data reported here are for laminar flow. Subconfluent BAEC cultures continuously exposed to 1-5 dynes/cm2 shear proliferated at a rate comparable to that of static cultures, and postconfluent monolayers appeared unaltered morphologically for up to 1 week. In contrast, BAEC cultures (both postconfluent and subconfluent) exposed to 8 dynes/cm2 developed dramatic, time-dependent morphological changes. By 48 hrs, cells uniformly assumed an ellipsoidal configuration, with their major axes aligned in the direction of flow. Exposure to >10 dynes/cm2 caused variable cell detachment from plain glass substrates. Cellular migration into linear “wounds”, created in confluent areas, was influenced by both the direction and amplitude of applied shear. Exposure to 8 dynes/ cm2 induced functional alterations, including increased fluid (bulk phase) endocytosis, prostaglandin production and platelet reactivity. These observations indicate that fluid mechanical forces can directly influence endothelial cell structure and function. Hemodynamic modulation of endothelial cell behavior may be relevant to normal vessel wall physiology, as well as the pathogenesis of atherosclerosis and thrombosis.
Cerebrovascular Disease - Results With Antithrombotic Therapy : Some Observations
Authors: Sandok B A
Published: July 2018

Antithrombotic therapy is now widely used in the treatment of patients with ischemic cerebrovascular disease. Governmental approval will increase use and further convince many that the issues of therapy are now resolved. They are not. The following approved professional labelling guidelines serve to highlight the many clinical and investigational problems that remain:
(1) There is evidence that aspirin is safe and effective for reducing the risk of stroke in men who have symptoms due to fibrin-platelet emboli.

(2) Patients should have a complete medical and neurologic evaluation.

(3) The recommended dosage is 1300 mgm/day.

The evidence for effectiveness, while of statistical significance, becomes clinically less important when one notes that in the Canadian study, a disturbing 12.4% of patients under treatment with aspirin, developed subsequent stroke. This should not be unexpected since similar cerebral ischemic symptoms may be produced by differing pathophysiologic mechanisms (not all related to fibrin-platelet embolization); and treatment for one may be illogical and ineffective for another. Accurate diagnosis is imperative for selecting appropriate therapy. In the clinical situation, in spite of “complete” evaluation, uncertainty of the underlying mechanism often remains. Questions also remain about the dosage recommendations, and other treatment alternatives for both women and men.

Subsequent clinical trials will need to consider each of the above. Until then, patients with cerebral ischemic symptoms must be evaluated individually. Some may benefit from antithrombotic therapy-many will not.
Does The Treatment Of Stroke Risk Factors Reduce The Incidence Of Stroke?
Authors: Conomy John P
Published: July 2018

The answer to the question posed in this title is “yes”, but affirmation is highly qualified. (1) Stroke is not a homogeneous clinical disease state but a dynamic process whose etiologies are manifold. Its causes vary from the commonplace atherothrombotic occlusion of brain arteries and emboli of cardiac origin to rare and complex precipitants. (2) Stroke is not a hemogenous pathologic state. Parenchymal hemorrhage, hemorrhagic infarctions and bland infarctions are very different forms of brain destruction with differing outcomes. The great triumvirate of risk factors in stroke are advanced age, arterial hypertension and heart disease. All of these conditions tend to coexist with the process of atherosclerosis. Hypertension and cardiac disease are potentially amenable to early diagnosis and treatment. In clinical studies, the effective treatment of arterial hypertension is emerging as the clearest evidence that risk factor modification is correllated with a decline in stroke. Therapy for some cardiac diseases constitutes effective stroke prevention as well. Other less direct risk factors for stroke tend to parallel the severity and extent of the atherosclerotic process. These include diabetes mellitus, hyperlipidemias and obesity. Therapeutic modification of these factors has debatable influence on the incidence of stroke and may reflect the atherosclerotic process to medical intervention. Some clinical studies have suggested that certain antiplatelet agents postpone stroke and limit TIAs, at least in males. Anticoagulants may prevent TIAs but do not appear to prevent stroke. Surgery has a definable range of risks and clearly demonstrated benefits in properly selected patients in whom it may relieve TIAs and postpone a given stroke. Surgery does not alleviate an individual patient’s overall stroke risk. The process of stroke prevention will remain imperfect until therapies for hypertension and heart disease are accompanied by more effective treatment of atherosclerosis.
The Structure Of The Fibrin Protofibril
Authors: Fowler W E , Erickson H P , Hantgan R R , Hermans J
Published: July 2018

We have identified the fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and considerations of symmetry we have constructed a model of the protofibril, in which two strands of the trinodular fibrin molecules are related by a two-fold screw axis between the strands and two-fold axes perpendicular to them. The molecules in a strand are joined by longitudinal contacts between outer nodules, and the two strands are held together by a single type of lateral contact between a central nodule on one strand and an outer nodule on the other. This interpretation of the structure of protofibrils is supported by images of trimer complexes, which show a single molecule of one strand attached laterally (by its central nodule) to the outer nodules of two molecules of the other strand. These lateral contacts, which are presumably the site of thrombin activation, are entirely saturated within the protofibril. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules of adjacent, inregister strands.
Different Affinity Of Fibrinogen To Des-A Fibrin And Des-Ab Fibrin At 37°C
Authors: Müller-Berghaus G , Thiel W , Mahn I V
Published: July 2018

To investigate fibrin-fibrinogen interactions at 37d°C, affinity chromatography was performed on fibrinogen-sepharose as well as fibrin-sepharose. As thrombin cleaves fibrinopeptides A and B from fibrinogen producing des-A fibrin and des-AB fibrin respectively, experiments were performed with both types of fibrin. Thrombin-induced, urea-solubilized des-AB 125I-fibrin as well as batroxobin-induced d es-A 125I-fibrin were mixed with plasma containing 131I-fibrinogen. These mixtures were passed through fibrinogensepharose columns equilibrated with tris buffer,pH 7.4, containing EDTA, benzamidine, aprotinin and NaN3 . The elution buffer (buffer B) contained tris and guanidine, pH 4.1. Results: At 37°C, des-A fibrin passed through the column together with fibrinogen, whereas des-AB fibrin stuck to the immobilized fibrinogen. In the next set of experiments, fibrin-fibrinogen mixtures were chromatographed on des-AB fibrin-sepharose . R e sults: At 20°C as well as at 37°C, des-A fibrin as well as des-AB fibrin were eluted together with fibrinogen with buffer B. In the last set of experiments, fibrin-fibrinogen mixtures were chromatographed on des-A fibrin-sepharose columns. Results: At 37°C, des-A fibrin as well as des-AB fibrin interacted with immobilized des-A fibrin, whereas fibrinogen did not stick to des-A fibrin. The results suggest that des-AB fibrin interacts with fibrinogen at 37°C. An interaction, if any, between des-A fibrin and fibrinogen is so weak at 37°C that it cannot be detected by affinity chromatography.
Localization Of A Fibrin Polymerization Site
Authors: Olexa Stephanie A , Budzynski Andrei Z
Published: July 2018

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B, and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur through the interaction of complementary binding sites on the NH2- and COOH- terminal regions of the molecules since Fragment D1, encompassing the COOH-terminals of the β and γ chains, binds to thrombin-treated NDSK which contains N2-terminals of the α , β and γ chains. A peptide of 4,200 molecular weight has been isolated from the y chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2- terminal region of fibrin monomer, thus inhibiting fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time of the reaction. The D1 peptide does not interact with NDSK, fibrinogen or Fragment D1but it binds to thrombin-treated NDSK with a Kd of 1.45 x 106 m and approximately two binding sites per molecule of NDSK have been found. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2- terminal amino acid sequence (Thr-Arg-Trp) and the COOHterminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the γ 373-410 region of the fibrinogen molecule contains a polymerization site which is complementary to the thrombin-activated site on the NH2terminal region of fibrinogen. The binding site on the Dl peptide has the characteristics of the polymerization site which is exposed and available on the COOH-terminal region of the fibrinogen molecule without any participation of thrombin.
Biological Activities Of Two Plasmin Preparations: Differential Binding To Fibrin And α2-antiplasmin
Authors: Philo R D , Gaffney P J
Published: July 2018

The intention to replace the present International Reference Preparation (IRP) for plasmin (a preparation auto activated from plasminogen in glycerol) with a freeze-dried urokinase (UK-activated plasmin (PL)) has prompted a comparison of the interactions of these two plasmins with fibrin and α2-antiplasmin (α2_APL). On a molar basis UK-PL was shown to digest fibrin about twice as rapidly as IRP-PL and these differential rates of fibrin lysis are reflected by the different binding affinities of these plasmins for forming fibrin.

The titration curves of plasma α2-APL with these two plasmins were constructed from data obtained when the residual PL was measured both by S-2251 and fibrin hydrolysis. With increasing amounts of plasma α2-APL, both plasmins were neutralised to near completion when a clot lysis assay was used. However when S-2251 was used the IRP plasmin was only about half neutralised.

The titration curve obtained when plasmin was neutralised with plasma α2-APL was similar in shape to an exponential decay whereas that for the reaction of purified α2-APL with UK-PL was a straight line. The IRP-PL reaction with purified α2-APL could be analysed as two straight lines.

The differential binding of these plasmins to fibrin and α2-APL suggested that the IRP plasmin is made up of at least two populations of plasmin molecules. The exponential binding curve found with plasma α2-APL could be explained by the presence of the histidine-rich glycoprotein in plasma which is known to inhibit the plasmin-antiplasmin reaction.

Polyacrylamide-detergent gel electrophoresis was used to 2onfirm the differences in functional plasmin/α2-APL data for the UK- and IRP-plasmins. These data could imply that a population of ‘plasmin’ molecules exists in the IRP which, because of the loss of certain strategic kringle regions in the heavy (A) chain, cannot bind to either fibrin or α2-APL.
Kinetics Of Plasminogen Activation By Tissue Plasminogen Activator. Role Of Fibrin
Authors: Hoylaerts M , Rijken D C , Lijnen H R , Collen D
Published: July 2018

The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.

According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.

Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.
Binding Of Blood Plasminogen Activator To Fibrinogen, Fibrin Monomer And Fibrin
Authors: Libeskind I C , Lipinski B , Gurewich V
Published: July 2018

It has been shown previously that plasminogen activator (PA) causes a conversion of plasminogen to plasmin in blood only in the presence of fibrin (F). By contrast to other activators (UK, SK) fibrinogenolysis is not initiated by PA in unclotted blood. In order to determine substrate specificity for PA binding and importance of a solid phase, the following experiments were performed. Human fibrinogen (FBG) was insolubilized either by binding to CNBr-activated agarose (FBG-AG) or by mixing with celite (1:20 w/w) and heating 3 min at 56°C. We have shown that celite prevents gel formation during clotting or heating of FBG and thus provides a rigid bed matrix for column chromatography. Part of FBG-AG was converted to insolubilized fibrin monomer by treatment with thrombin. Celite coated with polymerized F was prepared by treating with thrombin a mixture of FBG with celite (1:20 w/w). Columns (2ml vol) were packed with each material and equilibrated with phosphate buffer, pH 7.4, containing 0.3 M NaCl, 1 mM EDTA and 5 mM EACA. After passing 6 ml samples of fresh plasma, columns were washed extensively and eluted with the same buffer containing 0.2 M arginine. Binding of PA was determined as a percentage of activity in the arginine eluate as measured on standard fibrin plates. It was found that 5070% of PA present in human blood plasma binds to any form of insolubilized FBG or F. By contrast, gel filtration (Sephadex G-200) of a mixture of FBG and PA under identical conditions resulted in a separation of protein from the fibrinolytic activity peaks. It is concluded that blood PA has a specific affinity to insolubilized forms of FBG and that this may be a mechanism responsible for the absence of fibrinogenolysis in states of naturally activated blood fibrinolysi.
Serum Myoglobin In Acute Myocardial Infarction: Conservative Treatment Versus Acute Revascularisation By Intbacoronaby Streptokinase
Authors: Kaiser H , Sold G , blanke H , Schrader J , Rentrop P , Kreuzer H
Published: July 2018

Serial serum myoglobin (SMb) and CK were determined m 20 patients with angiographically proven acute myocardial infarction due to acute occlusion of a major coronary artery branch. In 10 patienus treated medically SMb nealied 10 hours before CK, both peal: values and total release correlated with percent akinetic segment in the chronic stage. 10 patients had effective revascularisation by intracoronary application of streptokinase. Here CK and SMb peaked earlier with SMb reaching its maximum 6.5 hours before CK; both maxima were higher than in the conservatively treated group. There was no linear correlation between CK and SMb oeak or CK and SMb release and extent of akinetic area indicating an improved wash-out from the infarcted area. Interrelations between jeopardized area reperfusion time and delay, and myoglobin release are discussed. In all these cases SMb seems to be the best biochemical parameter indicating successfull reopening of acute occluded coronary arteries by intracoronary thrombolysis wi th streptokinase.
The Effect Of Antiporcine Willebrand Factor Monoclonal Antibodies On The Bleeding Time
Authors: Bowie E J W , Fass D N , Katzmann J A
Published: July 2018

Murine monoclonal antibodies to porcine Willebrand factor were used to study the role of Willebrand factor in hemostatic plug formation in the in vivo and in vitro skin bleeding times. The in vivo assay requires the intravenous injection of antibody-containing mouse ascitic fluids into normal animals with subsequent ear bleeding time tests performed over a 24-hour period. The in vitro assay was carried out with heparinized normal porcine blood flowing through a 0.5 cm incision in an excised piece of porcine skin. The heat inactivated ascitic fluids, containing the antibodies, were used ;Ln these assays at final dilutions of 2 × 102 through 2 × 105 The 7 antibodies used comprised at least 5 groups with differing reactivities based on assays other than bleeding time tests. The antibody titers in a poreineg Willebrand factor-binding radioimmunoassay ranged from 108 through 1012 . Doses of 10HL of ascitic fluid/kg of body weight (approximately 2 × 103 final dilution) were able to transiently prolong the in vivo bleeding time without alteration of other VIII complex values (VIII:C, ristocetin cofactor activity and Vlll-related antigen). Higher doses resulted in bleeding times similar to von Willebrand pigs (>15 min) immediately following infusion, with a decay of the effect over the next 2 hours. At dilutions of 2 × 104 selected monoclonal antibodies interfered with hemostasis in the in vitro model whereas other antibodies inhibited only at 2 × 102 dilution. With one exception, the potencies of the antibodies appeared to be similar in both assays. The titer of the antibodies in the radioimmunoassay does not appear to correlate with prolongation of the in vitro bleeding time, suggesting specific reactivity with functional sites involved in hemostatic plug formatio.
Venous Volumetry In The Assessment Of The Post-Thrombotic Limb
Authors: Ruckley C V , Crawshaw H M , Seaton J
Published: July 2018

The reproducibility of venous volumetry has been evaluated in 20 normal legs (N), 20 legs with primary varicose veins (W) and 30 with stable post-thrombotic chronic venous insufficiency (CVI).

The test has shown significant differences between the groups in mean relative expelled volume (EV rel, ccs): N 1.65 ± SD 0.66; W 1.01 ± 0.40; CVI 0.64 ± 0.39 and in half refilling time (T3 secs): N 23.9 ± 11.5; W 11.8 ± 7.4; CVI 3.6 ± 1.8. The percent coefficients of variation in tests repeated at intervals of up to a month were as follows.

The reproducibility of the test confirms its value as a non-invasive means of measuring the severity of postthrombotic venous disease and thus as a means of assessing response to treatment. Serial volumetric tests on 25 patients before and for up to 12 months after Linton operations for chronic venous insufficiency show significant early improvements in expelled volume. But in many of these patients the refilling time tends to remain short indicating residual valvular incompetence and the possibility of clinical relapse in the long term.