bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Regeneration Time In Diabetics With And Without Retinopathy
Authors: Tindall H , Paton R C , Zuzel M , McNicol G P
Published: July 2018

Platelet regeneration time was measured by a modification of the Stuart technique which depends on progressive reappearance of the capacity to synthesize malondial- dehyde (MDA) in response to thrombin stimulation following aspirin ingestion. The subjects of the study were 40 normal subjects and 40 patients with insulin-dependent diabetes mellitus for more than 10 years. The presence or absence of retinopathy was independently assessed in a diabetic eye clinic and severe retinopathy was found in 20 patients. The mean platelet regeneration time in normal subjects was 9.98 ±0.22 days (mean ± SEM). In diabetic patients without retinopathy the platelet regeneration time was significantly (p<0.001) shortened (7.38 ± 0.14 days) and a further shortening occurred in those patients with retinopathy (6.80 ± 0.18 days), but with the number of subjects studied the difference between these two groups was not statistically significant. MDA produced in response to thrombin-stimulation of platelets was 235.5 ± 12.8 pmole/108 platelets in controls with significantly (p<0.001) less in diabetics with retinopathy at 156.7 ± 17.5 pmole/108 platelets. In 10 diabetic patients a standard oral glucose tolerance test was performed with blood taken for MDA at the same time as plasma glucose levels. There was no correlation between plasma glucose and the concentration of MDA produced. These results show that patients with longstanding insulin-dependent diabetes have an accelerated platelet regeneration time even in the absence of overt retinal microvascular complications.
Change In TXB2, MDA And AMP Levels During Artificial Pancreas Treatment On Diabetic Patients
Authors: Braun G , Guimont C , Voisin Ph , Rousselle D , Drouin P , Stoltz J F
Published: July 2018

A study carried out on 9 insulin-dependent diabetic patients undergoing continuous blood glucose control by means of ex-vivo insulin injection (artificial pancreas - Biostator) has revealed : a relationship between the synthetic capacity of TXA2 (proaggregating molecule) by the platelet and the blood glucose level prior to feed back control. Concurrently, an identical relationship is observed in the case of platelet AMPc levels.

After 24 hours of artificial pancreas treatment, it is noted that the greater the effectiveness of the treatment, the greater the increase in the synthetic capacity of TXA2. This observation is confirmed after 48 hours of treatment.

Further, the relationship between MDA and Thromboxane B2 synthetized from the same precursory molecules is reversed during treatment.

On the basis of these results we may assume that there is a disturbance in the regulation mechanisms of platelet synthesis during diabetes, but that these mechanisms are parthy reversible after artificial pancreas treatment.
Human Protein C, Its Inhibitor, And The Combined Deficiency Of Factors V And VIII
Authors: Marlar Richard A , Griffin John H
Published: July 2018

Human protein C (PC) is a vitamin K-dependent serine protease zymogen present in normal plasma. PC was activated with soluble thrombin, thrombin-Sepharose, trypsin-Sepharose or Russell’s viper venom. When activated protein C (APC) at 10 μg/ml was added to normal plasma for 3 min prior to assays, clotting was greatly inhibited. In prothrombin time, activated partial thromboplastin time, and partial thromboplastin time assays, the clotting times were prolonged from 24 to 68 sec, 57 to 616 sec, and 247 to > 1200 sec, respectively. Both calcium and phospholipid were needed for this potent anticoagulant activity. The thrombin time was unaffected. To test which coagulation factors in plasma were inactivated by APC, APC was added to normal human plasma for 3 min and residual clotting factor activities were assayed using appropriate deficient substrate plasmas. Only Factor V and Factor VIII activities were decreased by APC. PC and DFP-inhibited APC were not anticoagulant and did not decrease Factors V and VIII. APC hydrolyzed synthetic peptide substrates. The amidolytic activity of APC was inhibited by normal human plasma (50% of APC activity was lost in 10 min). APC inhibitory activity was not detectable in plasmas from 4 unrelated patients with combined Factor V/VIII deficiency whereas it was present in normal amounts in plasmas from patients with simple Factor V deficiency or Factor VIII deficiency. Thus, it appears that APC may regulate blood coagulation by enzymatically inactivating two critical cofactors, Factor Va and Villa. Moreover, APC itself may be regulated by a previously undescribed plasma protein inhibitor. It is suggested that the molecular basis for combined Factor V/VIII deficiency disease that exhibits simple autosomal recessive inheritance is a deficiency of APC inhibitor.
Regulation Of Bovine Activated Protein C By Protein S: The Role Of The Cofactor Protein In Species Specificity
Authors: Walker F J
Published: July 2018

The anticoagulant activity of activated Protein C has been observed to be species specific. This could be due either to the inability of the bovine enzyme to recognize its substrate, Factor Va, in non-bovine plasmas, or the absence of cofactor-Protein S, a protein that has been shown to be necessary for the maximum expression of the anticoagulant activity of activated Protein C. Activated Protein C was found to be an effective inhibitor of Factor Xa-initiated clotting of bovine plasma, but without activity in either human or rabbit plasma. Human and rabbit plasma supplemented with bovine Protein S was sensitive to the anticoagulant activity of activated Protein C. Neither rabbit nor human plasma contained bovine activated Protein C cofactor activity as measured by the enhancement of bovine activated Protein C-catalyzed inactivation of Factor Va. However, bovine activated Protein C was able to inactivate both human and rabbit Factor Va. The inactivation of both of these proteins could be stimulated by the addition of bovine Protein S. These results indicate that the species specificity of bovine activated Protein C is due to the absence of a cofactor protein in non-bovine plasma that will interact with the bovine enzyme. Secondly, these findings further confirm that Protein S is required for the maximal expression of the anticoagulant activity of activated Protein C.
Role Of The Fibrinolytic System In Renal Transplantation
Authors: Glas-Greenwalt P , Goldman M H
Published: July 2018

To determine the importance of the fibrinolytic system in renal transplantation on the one hand, and to establish a correlation between possible endothelial damage due to treatment of the renal graft and fibrinolytic parameters on the other, dogs were divided in six groups. Group I dogs were subjected to anesthesia only. Group II was sham operated. In group III, kidneys were perfused with the supernatant of either autologous or homologous cryo-precipitated plasma, and in group IV with albumin. In group V kidneys were cold stored. This was followed by autotransplantation. In group VI kidneys were perfused with albumin and allografted. Before and after transplantation, total plasma plasminogen (pro) activator activities in systemic and renal circulations were measured on fibrin plates after the addition of dextran sulfate and flufenamate to euglobulin fractions. Vascular activator (VA) was measured by adding Cl-inactivator. There was no marked difference between groups III, IV and VI. In comparing, however, group V with any of the perfused groups, an overall higher fibrinolytic activity was recorded both for intrinsic activators (p<.001) and VA (p<.001). in group I a significant drop in both activities (p<.01 -<.02) could be directly related to the duration of anesthesia with recovery within 24 hours. This pattern, though highly accentuated, revealed itself in all the other groups studied, with VA temporarily reaching zero-levels in the renal circulation in group VI. This data indicates the participation of the fibrinolytic system, in particular of the VA, in determining the fate of renal grafts.
Effect Of A Fish Oil On Blood Lipids And Coagulation
Authors: Saynor R , Verel D
Published: July 2018

Considerable evidence lias accumulated on the association between abnormal lipoprotein patterns and ischaemic heart disease. There is suggestive evidence that prolonged byper- lipidaemia may cause an increased tendency to thrombosis as a result of a number of factors. The Eskimo diet of marine animals is high in elcosapentaenoio acid (EPA) and it is possible that the low incidence of nyocardial infarction in this race is due to diet. The high intake of SPA by Eskimos is probably the reason for their increased tendency to bleed.

The present study was designed to observe the effectiveness of a fish oil high in EPA as a lipid-lowering agent, and its effect on coagulation. Of the 18 subjects in the study 6 were volunteers with no evidence of heart disease- the remaining 12 were attending hospital with angina or post-myocardial infarction. One of this group had undergone a coronary artery by-pass graft operation.

Venous blood was taken after a 12-14 hour fast and the subjects instructed to take 10ml of fish oil twice daily with meals. No modification of their normal diet was attempted. A further blood sample was taken 6 weeks later and at 3 months in 5 subjects. Laboratory investigations included blood cholesterol, triglyceride, high density lipoprotein cholesterol (CHDL;, bleeding time, platelet count and agpegation, antithrombin III, fibrinogen, thrombin time partial thromboplastin time and prothrombin ratio.

At six weeks the cholesterol fell lqy an average of 6.3% and triglyceride 36.8% but CHDL was raised 12.4%. The 3 month levels (5 subjects) were cholesterol - 9.2%, triglyceride - 52.1% and CHDL + 16.9%. There were no significant changes observed in the coagulation parameters measured.

From the foregoing it would appear that supplementing the diet with fish oil has a desirable effect on blood lipids but at this dose does not influence coagulation.
Antiplatelet Actions Of Low Molecular Weight Peptides And Their Derivatives
Authors: Fareed Jawed , Messmore Harry L , Walz Daniel A
Published: July 2018

We have reported on the antiserine protease actions of low molecular weight peptides with arginine and lysine at the carboxyl terminus and their derivatives. In order to investigate the action of these peptides and their analogues on other components of hemostasis we studied their effects on platelet function; aggregation and release reactions, clot retraction and serotonin uptake by platelets. D-Phe-Pro-Arg-NH-heptyl, D-Phe-Pro-Arg-O-heptyl, D-Phe-Pro-Arg-thiobenzyl, D-Phe-Pro-Arg-COOH, D-Phe-Pro-Arg-NH2, D-Phe-Pro-Arginal and similar derivatives of D-Pro-Phe-Arg, D-Phe-Phe-Arg and Val-leu-lys- were screened None of these peptides produced aggregation and release reactions in concentration >10 mM. D-Phe-Pro-Arg-thiobenzyl and D-Phe-Pro-Arginal produced a strong inhibition of thrombin induced aggregation and release reactions at sub ymolar levels. Both of these peptides also inhibited thrombin’s action in amidolytic and coagulant assays. D-Phe-Pro-Arg-thiobenzyl ester also produced a complete inhibition of arachidonic acid induced aggregation of platelets and showed varying inhibitory actions against ADP, epinephrine, collagen, and serotonin induced aggregation and release reactions. Although ristocetin induced aggregation was only slightly effected, a complete block of the release reaction was seen. ADP induced β-thrombo- globulin release was also inhibited by this peptide.

Heptyl amide and esters of H-D-Phe-Pro-Arg-and free acid, amide and arginal forms of various other peptides exhibited relatively weaker antiplatelet actions. Our studies suggest that peptides with amino acid resembling serine protease sensitive sites can be molecularly manipulated to design potent antiplatelet agents. Furthermore these peptides may provide a useful probe to study the molecular mechanisms involved in the pathophysiology of thrombotic disorders and to design new antiplatelet drugs.
Scanning Electron Microscopic Evaluation Of Dimethyl Sulfoxide, Barbiturate Or Steroid Antiplatelet Aggregation Properties
Authors: Dujovny M , Segal R , Kossovsky N
Published: July 2018

Despite advances in instrumentation, suture material, and operative techniques, thrombus formation at the anastomotic site remains a major factor in compromising surgical results in cerebral revascularization. Dimethyl sulfoxide (DMSO), methyl prednisone, thiopental, and pentobarbital are drugs that have been shown both clinically and experimentally to have protective effects in focal cerebral ischemia, and also to possess antiplatelet properties. We undertook a study on the carotid arteries of 72 Sprague- Dawley rats to assess the effect of the above drugs on microvascular thrombus formation. The control group consisted of 24 arteries, 12 that had an aneurysm clip applied for 1 hour, and 12 that had an arteriotomy and suture repair; no pharmacological agent was administered to this group. The experimental group was sub-divided into 2 major subgroups: Group A consisted of a total of 24 arteries subjected to aneurysm clip application for 1 hour; Group B consisted of a total of 24 arteries subjected to arteriotomy and suture. In both experimental groups, 6 animals were treated with one of the following pharmacological agents that was infused IV over a period of 1 hour: DMSO 2 mg/Kg; methyl prednisone 40 mg/Kg, thiopental 90 mg/Kg, pentobarbital 90 mg/Kg. The tissue was recovered 15 minutes after arterial segment reperfusion and prepared for Scanning Electron Microscopy (SEM). The specimens treated with DMSO, despite showing fracture of the endothelial layer with exposure of the sub-endothelium, had minimal thrombus formation. Specimens treated with the barbiturates showed less thrombus formation than the control group, but not comparable to the effect obtained with DMSO. The methyl prednisone group showed no difference compared to the control group.
Recovery Of Platelet And Endothelial Cyclooxygenase After Aspirin Measured By Radioimmunoassay For Thromboxane B2 And 6-Keto-Pg-F1α
Authors: Greaves M , Bull Helen , Castaldi P A
Published: July 2018

Recovery of platelet and endothelial prostaglandin synthetic activity by membrane cyclooxygenase after aspirin suppression of the enzyme is relevant to attempts to influence these pathways in platelet and microvascular disorders. Recovery from platelet inhibition has previously been measured by a colorimetric technique and proposed as a method for measurement of platelet turnover. Indirect methods have been used to determine prostacyclin production after similar inhibition and have not clarified the precise recovery rates. In the present study recovery in human and rat platelets was measured using a radioimmunoassay (RIA) for thromboxane B2 (TXB2) and in rat aortic endothelium with a RIA for 6-keto-prostaglandin- F1α (6KPGF1α).

TXB2 was measured in human platelet rich plasma stimulated with collagen and arachidonic acid before and after ingestion of 900 mg of aspirin. TXB2 returned to preaspirin levels in 8.5 ± 0.6 (S.D.) and 8.25 ± 0.5 days respectively with collagen and arachidonic acid, the recovery corresponding with platelet survival time of 51Cr autologous platelets. The recovery curve was linear with collagen but not with arachidonic acid where a late rise occurred.

Rats were treated with a single dose of aspirin 20 or 100 mg/kg and sacrificed at intervals for measurement of collagen stimulated platelet TXB2 aortic endothelial 6KPGF1α- Recovery of both of these products was linear and reached pretreatment levels after 120 hrs in platelets and 48 hrs in endothelium after 20 mgAg but 100 mg/kg delayed endothelial recovery to 96 hrs.

These results indicate that the endothelial and platelet enzyme are equally susceptible to aspirin but endothelial recovery is more rapid at low doses. Collagen stimulated platelet recovery may prove a useful method for measuring platelet production.
Effects Of Altered pH And Sodium Concentration On The Response Of Megakaryocytes And Platelets To ADP
Authors: Leven R M , Nachmias V T
Published: July 2018

By using both megakaryocytes and platelets to study effects of adenosine diphosphate (ADP), we hope to understand the ionic events underlying the shape changes induced by this nucleotide. Guinea pig bone marrow megakaryocytes were isolated to 50-90% purity by modifications of published procedures, and cultured for 12-48 hours. In response to ADP (l-10μM) megakaryocytes develop a ruffled border and spread in 15-30 minutes to 2-3 times their original diameter and adhere strongly to the substrate. Several other diphosphonuc1eotides or adenosine monophosphate have no effect. Spreading occurs in calciummagnesium free Hank’s salt solution, but is blocked when extracellular sodium was replaced by potassium, choline, or lithium. Amiloride, an inhibitor of sodium uptake, inhibits the extent of spreading 90% at 10-4M, but allows partial spreading in 60% of the cells. Spreading can be mimicked by incubating the cells with 2μM A-23187 combined with either 5mM methylamine or 1μM monensin, but not by A-23187 alone.

The rate of platelet shape change was measured spectro- photometrical1y at 37°C with 5mM EGTA both in platelet rich plasma (PRP) or after gel filtration in phosphate buffered saline. Amiloride inhibits shape change completely at 1mM and 50-60% at 0.1mM, but only if present during gel filtration. A rapid drop in buffer pH from 7.4 to 6.9 causes a 50% inhibition of rate of shape change in PRP or after gel filtration, but the inhibition spontaneously reverses within three minutes incubation at the lower pH.

These results are compatible with a model in which ADP causes a sodium influx which is linked to a change in intracellular pH. This may be a necessary but not necessarily sufficient step in ADP induced shape change.
Cerebrovascular Injuries Caused By Intravascular Platelet Aggregation Induced By Arachidonic Acid (AA) Or ADP
Authors: Yamazaki H , Fugimoto T , Kobayashi M , Matsubara O , Tanoue K , Isohisa I , Inaba Y
Published: July 2018

Effects of acutely induced intravascular aggregation of platelets on cerebral arteries were examined. A polyethylene catheter was inserted into the right lingual artery through the right carotid artery. A group of 30 rabbits (22 with no pretreatment , 8 thrombocytopenic) were given 0.7 mg/kg of AA as a bolus within 5 sec through the catheter. Thirty min. later, Evans blue was injected as marker for extravasation. Another group of 20 rabbits (12 no pretreatment, 8 thrombocytopenic) were given 20 mg/kg of ADP within 1 min. One hour after injection, brains were observed histologically after perfusion of fixatives. Brains of 77%(17/22) of AA-injected rabbits showed irregular blue staining in the right hemisphere. Extravasation of plasmal substance was histologically observed in medium or larger-sized cerebral arteries. Frequent platelet adhesion on the luminal wall was seen. Scanning electron micrograms revealed almost all adherent platelets were spherical and aggregated. Fibrin formation was rarely seen. Derangement of endothelial lining was apparent around the area to where platelets adhered.

Some places showed irregular concaves and others a flattened luminal lining. Endothelial cells were lacking in some places. The changes were not observed in the opposite hemisphere or in the thrombocytopenic group. Sixty-seven percent (8/12) of ADP injected rabbits showed marked blue staining and edematous changes in the right cortex. Ipsilateral hemispheres were stained in 2 cases and 2 cases showed no changes. The ADP-injected group showed similar, but less pronounced histological changes. Platelet count was more marked and longer duration in the AA group as compared to the ADP group. Prominent decreases were seen in the cases which showed remarkable vascular changes. These vascular injuries may be related to the consumption of intravascular platelet aggregates. Activated platelets might play an important role in the genesis of vascular injuries.
Thrombin-Induced Refractoriness Of PGI2 And Adenine Nucleotide Release From The Vascular Endothelium
Authors: Brotherton A F Adams , Hoak J C
Published: July 2018

Primary monolayer cultures of human umbilical vein endothelium release both PGI2 (measured by radioimmunoassay of 6-keto-PGF1α) and adenine nucleotides (AN) (measured as release of 3H from monolayers prelabeled with [3H]adenosine) when incubated with thrombin (0.5 U/ml) or A23187 (10 μM) .The release of PGI2 and AN in response to A23187 was greater than that observed in response to thrombin by an average of 2- and 3.5-fold, respectively. Preincubation of the endothelium with aspirin (100 μM) for 30 min resulted in a total inhibition of thrombin or A23187 induced release of PGI2, but had no significant effect on the release of AN. Thus, AN release does not appear to be dependent on PGI2 biosynthesis. After an initial exposure to thrombin, the endothelium became refractory to both PGI2 and AN release. Prior exposure of the endothelium to thrombin also decreased A23187 induced release of PGI2 by an average of 50%, but had no effect on AN release. Preincubation of the endothelium for 10 min with the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (4 mM) increased the intracellular concentration of cyclic AMP (measured by radioimmunoassay) by an average of 3.5-fold, and caused a total inhibition of thrombin and A23187 induced release of PGI2 and AN. Although PGI2 (400 nM) and ADP (10 μM) increased cyclic AMP levels by several-fold in IBMX-treated endothelium, no significant increase was observed in the absence of IBMX. These findings suggest that the phenomenon of thrombin-induced refractoriness is not the result of a simple negative feedback mechanism involving the activation of adenylate cyclase by PGI2 and AN released from the endothelium. Complex mechanisms involving the thrombin binding mechanism, depletion of Ca2+ gradients or inactivation of Ca2+-dependent reactions are more likely to be responsible.
Localization Of Antithrombin III On The Vascular Endothelium And Its Function
Authors: Nakagawa M , Okajima Y , Kitani T , Watada M , Yoshikawa H , Hino R , Ijichi H
Published: July 2018

Vessel wall is known to be rich in sulfated glycosamino glycans (GAGs) including heparin and they may serve as the natural catalysts of antithrombin III (AT III). This paper reports the possible role of AT III on the development of anticoagulant activity of activated AT m on the vessel wall. The freshly prepared rat aortic ring was washed in 0.05M Tris HC1 0.15M NaCl buffer (ph 7.5) with or without protamine, and was fixed for histochemical and immunofluo- rescent examinations. Anti-rat AT III serum was obtained from rabbit immunized with the purified AT III. Mucopolysaccharide stainings including Alcian Blue revealed the sulfated GAGs on the endothelium and the media. The GAGs on the endothelium corresponded with the specific AT III fluorescence localization. Protamine treatment disclosed the disappearance of the AT III fluorescence on the surface of endothelium. AT III-heparin complex was observed to be dissociated by protamine treatment through the investigations with affinity chromatography and sephadex G75 column chromatography, and the separated AT III was proved to recover the original progressive antithrombin activity.

Based on these experimental results it is concluded that the localized AT III on the surface of endothelium may be present in complexed with the exposed heparin or other GAGs and be present in activated, and that the complexed AT III is considered to inhibit proteases in situ in the circulating blood to prevent the activation of coagulation process. This phenomenon may have a clinical significance on the pathogenesis and treatment of thromboembolic vascular diseases.
Endothelial Permeability And C-14- And I-131-Cholesterol Uptake On Aortocoronary Femoral Vein Bypass Grafts In Dogs
Authors: Dewanjee M K , Shapira N , Tago M , Fuster V , Kaye M P
Published: July 2018

Eighteen dogs underwent aortocoronary femoral vein bypass graft surgery. All dogs were injected with 300 μCi of I-131-iodocholesterol and 100 μCi of C-14-cholesterol and sacrificed 24 hours postoperatively: group I dogs, 1 day; group II dogs, 3 days; group III dogs, 7 days; and group IV dogs, 1 year after surgery respectively. Radioactivity in blood, grafted femoral vein (GFV), control femoral vein (CFV), femoral artery, aorta and vena cava were determined with Beckman gamma and beta counters. Results indicate a sixfold increase in permeability as assessed by uptake of cholesterol in grafted vein with respect to control vein in the 1-day period postsurgery.

The cholesterol uptake in GFV (p<.01) decreases to half of this value seven days after surgery. Scanning electron microscopy demonstrates patchy endothelial cell loss from GFV and slow re-endothelialization which decreases cholesterol uptake.

Vein graft permeability as assessed by cholesterol uptake thus provides a sensitive marker of graft de- endothelialization. This information is now permitting us to better understand the platelet-arterial wall interaction and of the role of platelet inhibiting drugs at different stages after aortocoronary bypass surgery.
Isolation And Characterization Of Platelet Membrane Glycoprotein IIB-IIIa With A Monoclonal Antibody
Authors: McEver R P , Baenziger N L , Majerus P W
Published: July 2018

We have developed hybridoma cell lines that produce monoclonal antibodies directed to different components on the human platelet membrane. One of these lines produces an antibody, named Tab, that binds to a protein on normal platelets that is deficient in platelets from patients with Glanzmann’s thrombasthenia. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-agarose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein consists of two polypeptides previously designated as glycoproteins IIb and IIIa. The isolation of two polypeptides by a single monoclonal antibody suggests that IIb and IIIa are subunits of a single molecule. We prepared 125I-Tab to determine the number of glycoprotein IIb-IIIa molecules on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal individuals bound 39,000 ± 4,600 Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%) . Nine heterozygotes for thrombasthenia bound 24,500 ± 5,800 Tab molecules/platelet. We separated the IIb and IIIa subunits by preparative gel electrophoresis and analyzed their structures. Both polypeptides contain ∼15% carbohydrate, but IIIa has a higher percentage of mannose residues. The amino acid compositions are similar, suggesting some sequence homology. However, tryptic peptide maps of 125I IIb and 131I IIIa, analyzed simultaneously by high performance liquid chromatography, are completely different. This indicates that neither subunit is derived from the other and suggests that IIb and IIIa may be products of separate genes. The Tab antibody should prove useful in further elucidation of the structure and function of glycoprotein IIb-IIIa. Other monoclonal antibodies may serve as valuable probes of additional platelet membrane proteins.
Antigenic Properties And Structure Of Membrane Glycoprotein Receptors
Authors: Kunicki T J
Published: July 2018

Membrane glycoproteins (GP) IIb and IIIa are not detectable on the platelets of a majority (Type 1) of patients with Glanzmann’s thrombasthenia, a hereditary disorder of platelet function characterized by an absence of platelet-platelet adhesion. These platelets also do not express the human platelet alloantigen, PlA1, a finding which led to the demonstration that the PlA1 alloantigen is associated with GPIIIa, but not with GPIIb. Alloantibodies directed against the PlA1 determinant or certain other determinants associated with GPIIb and/or GPIIIa appear to be capable of inhibiting normal platelet- platelet adhesion, suggesting that these glycoproteins function as key mediators of this process, an obligate step in normal hemostasis. Subsequent studies have supported this hypothesis by demonstrating that fibrinogen, which is known to be a key cofactor in normal platelet aggregation, does not bind to thrombasthenic platelets.

We have used crossed immunoelectrophoresis of Triton X-100 soluble membrane glycoproteins against a multispecific rabbit antibody to quantitate levels of GPIIb and GPIIIa on normal and thrombasthenic platelets and to study the interaction of these two glycoproteins in a soluble nondenatured state. These studies have provided information on 1) the inheritance of PlA1 and the glycoprotein IIb/IIIa defect in Glanzmann’s thrombasthenia, and 2) reversible Ca++-dependent changes in the orientation of GPIIb and GPIIIa, which may contribute to understanding of the function of these glycoproteins in normal platelet aggregation. In the presence of sufficient Ca++, glycoproteins IIb and IIIa were found to exist only in heterogenous complexes, whether within isolated membranes, or in solution. Chelation of Ca++ by EDTA or EGTA resulted in the dissociation of these glycoproteins; reassociation could be induced by readdition of excess Ca++.
The Vascular Smooth Muscle Cell In Culture
Authors: Campbell G R , Chamley-Campbell J H
Published: July 2018

When smooth muscle from the aortic media of adult pigs, monkeys and rabbits are dispersed into single cells and seeded into primary culture they are in the contractile phenotype and do not undergo cell division when challenged with 5 or 10% whole blood serum. However, after 6-8 days the isolated cells undergo a spontaneous change in phenotype to a synthetic state whereby their contractility is lost and they become responsive to serum mitogens. This change in function is accompanied by distinct morphological changes and is reversible if the cells are seeded sufficiently dense (5 × 104 -1 × 105/ml) that a confluent monolayer of cells is achieved within 5-7 days of the original change in phenotype. If however the cells are seeded so sparsely (1 × 103 - 5 × 103/ml) that 2-3 weeks of proliferation is necessary for confluence to be achieved then the cells remain permanently in the synthetic phenotype and immediately responsive to serum mitogens upon subculture. While the cells are in the synthetic phenotype, whether proliferating or quiescent, they have a considerably decreased production of prostaglandins E2, F2α and I2. They also have a decreased ability to metabolize low density lipoprotein, a decrease in the specific activity of cholesteryl esterase and an increase in cholesterol ester deposits.

The spontaneous change in phenotype from the contractile to synthetic state in culture can be prevented by seeding the contractile smooth muscle sufficiently dense (1 × 106/ ml) that a confluent monolayer is present from day 1. It can also be inhibited by a confluent monolayer of contractile smooth muscle or endothelial cells in co-culture with the sparsely-seeded smooth muscle such that the two cell layers are not in contact but bathed by the same nutrient medium. A factor which inhibits smooth muscle phenotypic change can also be extracted from pig and rabbit aortic tissue and its effect mimicked by sodium heparin at 50 units/ml.

It is suggested that smooth muscle phenotype and its control are important but unrecognized factors in atherogenesis.
Growth Regulation Of Endocytosis In Cultured Vascular Cells
Authors: Davies Peter F
Published: July 2018

Endocytosis is involved in the metabolism of blood-borne macromolecules by both vascular endothelium and smooth muscle cells (SMC). Intimal SMC proliferation is a prominent feature of atherogenesis and therefore the interactions between the cell cycle, growth factors and endocytosis are relevant to this disease process. Manipulation of the growth of smooth muscle cells in culture by platelet- derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) has demonstrated a coupling between endocytic volume (fluid endocytosis), endocytosis of low density lipoproteins (LDL) and growth regulation. Fluid endocytosis rate increased up to 10 fold per responding cell early in the cell cycle and continued elevated through S phase before declining. The magnitude of the effect was proportional to the fraction of cells entering the cycle, and when density-dependent inhibition of growth occurred, it was accompanied by density-dependent inhibition of fluid endocytosis rate. In addition to these changes in rate of fluid endocytosis, we also studied the high affinity binding, endocytosis and degradation of LDL. PDGF and FGF stimulated LDL binding by up to 300% and this also resulted in enhanced LDL degradation. Removal of growth factor after initiation of the cell cycle, however, abolished the response, suggesting that the continued presence of growth factor was required for enhanced LDL binding.

A direct relationship between fluid endocytosis and cell growth was also measured in cultured endothelium, but exposure of confluent endothelium to PDGF or FGF resulted in increased binding of LDL without a growth response.

These results, together with the failure to enhance binding of LDL to cycling SMC after removal of mitogen, suggest that growth factor stimulation of LDL binding and its consequences may be uncoupled from cell growth even though the common carrier for internalisation (endocytic vesicle volume) is closely related to the cell cycle.
Arterial Injury Alters Metabolism Of The Arterial Wall
Authors: Minick C R , Hajjar D P , Falcone D J , Fowler S , Eldor A , Weksler B B
Published: July 2018

Results of our previous experiments indicate that following de-endothelialization aortic intima was thicker and contained more lipid in re-endothelialized (RA) and not de-en- dothelialized areas (DA) as we anticipated. Chemical analysis revealed that, in rabbits fed lipid-poor diets, there was 2 to 3 times as much cholesteryl ester (CE) in RA as compared to DA or uninjured aortas. In hypercholesterole- mic rabbits, aortic total cholesterol content correlated with serum cholesterol concentration in RA but not DA.

Taken together, these findings suggest that aortic cholesterol (CH) and CE accumulation are not simply a result of increased filtration of lipoproteins into the de-endo- thelialized aorta but may result from altered metabolism of the arterial wall. Previous findings showing increased glycosaminoglycan (GAG) accumulation in RA supported this concept of altered metabolism and suggest that these GAGs may sequester lipoproteins. Recent experiments indicate that: 1) CE synthetic and hydrolytic activity as well as activities of several marker enzymes were increased in injured aortas, 2) CE hydrolytic activity was relatively greater in DA as compared to RA, 3) Neointimal smooth muscle cells (SMC) in DA synthesized increased quantities of PGI2 and that PGI2 increased CE hydrolase activity of SMC and 4) RA accumulate more 125I-apo-B-lipoproteins than adjacent DA. In conclusion, results indicate that RA accumulate more CE rich apo-B-lipoproteins and have decreased ability to degrade CE. This may, at least in part, account for the increased CH and CE accumulation in RA. Further, the findings suggest that arterial injury alters arterial wall metabolism and that interactions between neointimal smooth muscle cells and endothelial cells may modify these metabolic changes.
The Role Of Platelets In Acute Vascular Events
Authors: Harker L A
Published: July 2018

Platelets are postulated to have an important role in acute and chronic cardiovascular events. Clinical events may result during the sequence of platelet activation reactions of adhesion, aggregate formation, release of granular constituents, thromboxane A2 formation, microembolization and platelet vascular occlusion. For example, sudden death may occur in patients with increased platelet sensitivity to catecholamine stimulation, platelet aggregate formation and thromboxane A2 generation resulting in ischemic ventricular arrhythmia induced by small-vessel platelet microembolization and local thromboxane A2 vasoconstriction. Whereas angina might be manifest if the microemboli disaggregated and vasospasm were transient, myocardial infarction would follow extensive permanent occlusion of small vessels or localized narrowing by vasospasm and extension of platelet thrombus formation at the site of intimal thickening. Platelets may also have an important role in atherogenesis through the mediation of the platelet-derived growth factor in the proliferative smooth muscle cell intimal lesion. Direct experimental and indirect clinical studies support the concept that platelets are important in cardiovascular events, but the relationship to spasm and to other possibly even more important mechanisms is not clear. Acute myocardial infarction and mural thrombogenesis appear to have the greatest evidence of being platelet-related. Proof that platelets play a critical, primary role in coronary artery disease and its complications ultimately requires a successful clinical intervention with platelet modifying agents. Although there are positive aspects of several controlled studies with platelet inhibiting agents, the secondary intervention trials have so far been inconclusive.
Overall Structure Of The Heparin Molecule
Authors: Atkins Edward
Published: July 2018

The polysaccharide suppresses the coagulation of blood by controlling the rate at which the plasma protein antithrombin inactivates the proteases of the coagulation cascade. One facet necessary in the full understanding of the interaction of heparin with antithrombin is the detailed shape of these macromolecules at an atomic resolution. X-ray diffraction methods are necessary to obtain the required information since the wavelength of the radiation is comparable with the distances between atoms. Analysis of the diffraction signals coupled with computerised model building and energy minimization procedures enables the three dimensional structure, or conformation, of the heparin molecule to be examined. A major part of the molecule is a repeating disaccharide of alternating 1.4-linked-2-deoxy-2-sulphamino-α-D-glucose-6-sulphate and 1.4-linked 2-sulphate-α-L-idopyranoslyluronic acid. This is a first order statement, made necessary to avoid confused and sometimes wrongful interpretations of past chemical analyses (which for many years suggested β-D-gluco-pyranosyluronic acid on the prominent uronic acid in heparin). More recent chemical analyses of selected heparins exhibiting high anticoagulent activity indicates that the idealised polydisaccharide repeat is perturned by local variation in the chemistry. The absence of a sulphate appendage on iduronic acid and the replacement of iduronic acid by glucuronic acid in the near vicinity are features proposed. The effect of these chemical modifications on the local conformation of the heparin molecule is considerable and suggest a specific binding site region within the heparin macromolecule amenable to interaction with antithrombin.
The Heparin Binding Site Of Antithrombin: Identification Of A Critical Tryptophan Residue Within The Amino Acid Sequence
Authors: Blackburn M
Published: July 2018

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.
Growth Factors Produced By Cultured Human Vascular Endothelial Cells
Authors: Willems Ch , Astaldi G C B , de Groot Ph G , Janssen M , Gonsalvez M D , Zeijlemaker W P , van Mourik J A , van Aken W G
Published: July 2018

The growth of cells in culture is regulated by several as yet undefined growth factors which are normally present in serum and released by cultured cells in vitro. We have investigated media conditioned by cultured vascular human endothelial cells (HEC) and smooth muscle cells (SMC) derived from umbilical veins and expiants from umbilical cords respectively. Cell proliferation was measured by 3H thymidine uptake. The conditioned medium of confluent endothelial cells (HECS) contain(s) oneor more factors which inhibit the growth of nonconfluent endothelial cells and SMC. HECS derived from actively growing HEC as well as conditioned SMC medium were inactive. The growth factor(s) is (are) also present in serum free HECS and is released in a time dependant fashion at least up to 72 h. The release of HECS does not correlate with cell lysis, but our experiments indicate that protein synthesis is required for the generation of HECS. HECS contains also potent growth factor(s) for hybridoma cells particularly for xenogenetic hybridomas between mouse myeloma cells and human lymphocytes. HECS can substitute for feeder cells in cloning by limiting dilution of hybridoma cells. The growth of hybridoma cells is enhanced, both by a 24 h “pulse” with HEC as well as in its continuous presence during culture, indicating that the effect is based on delivering a growth signal to the cells. In preliminary experiments to characterize HECS, the following findings were observed, - HECS production requires protein synthesis in endothelial cells, - the activity is abolished by treatment with periodate, suggesting that sugar moieties are involved, - on gel filtration HECS activity is associated with a molecular weight of 20-30×103. These findings demonstrate that one or more growth factors are synthesized and released by vascular endothelial cells, a finding which underlines once more the pivotal role of the endothelium in biological systems.
An Inhibitor Of Plasminogen Activator In Rabbit Endothelial Cells
Authors: Loskutoff David J , Edgington Thomas S
Published: July 2018

The anti fibrinolytic activity of cytosols obtained from cultured rabbit endothelial cells was studied to determine whether it resulted from the presence of an antiplasmin or an antiactivator. These cytosol preparations inhibited the fibrinolytic activity initiated by some plasminogen activators (e.g., urokinase, rabbit endothelial cell activator), but not others (e.g., activators associated with bovine endothelial cells and Rous sarcoma virus infected chick embryo fibroblasts), suggesting that inhibition occurred at the level of plasmin formation, not plasmin activity. The fibrinolytic activity of plasmin itself was unaffected by concentrations of cytosol that completely blocked urokinase-mediated fibrinolysis, consistent with this conclusion. In addition, the ability of urokinase to cleave 125I-plasminogen into its characteristic activation fragments was inhibited by. cytosol in a dose-dependent manner. When urokinase was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two peaks of activity were detected, corresponding to molecular weights of 55,000 and 32,000. Urokinase preincubated with cytosol and analyzed in a similar manner demonstrated no activity in any portion of the gel, suggesting that its ability to function as a plasminogen activator was irreversibly lost following its interaction with cytosol. These results indicate that the anti fibrinolytic activity of rabbit endothelial cells results from the presence of an anti activator. The cellular location and unusual degree of specificity distinguish the endothelial cell inhibitor from antifibrinolytic agents observed in other cells and in plasma and platelets.
Measurement Of Prostaglandins That Activate Platelet Adenylate Cyclase In Rabbit Arterial Blood; Studies On PGI2 And 6-KETO-PGE1
Authors: Haslam R J , McClenaghan M D
Published: July 2018

The increases in platelet cyclic [3H]AMP on addition of washed rabbit platelets containing 3H-labelled adenine nucleotides to fresh arterial blood samples mixed with 1 mM 3-isobuty1-1-methyl xanthine permitted assay of any activators of adenylate cyclase present. Blood PGI2 was calculated from the difference between the increases in cyclic [3H]AMP over 30 s in the presence and absence of antibody that bound PGI2 and the effects of PGI2 standards on cyclic [3H]AMP formation in preincubated blood. The PGI2 found in rabbit blood assayed within 2.5 min of arterial puncture (0.05 ± 0.01 pmol/ml in 10 males and 0.07 ± 0.02 pmol/ml in 8 females (means ± S.E.M.)) was far less than that needed to inhibit platelet function (>1 pmol/ml). The antibody used also bound 6-keto-PGE1, a metabolite of PGI2 that was one fifth as effective, both as an activator of rabbit platelet adenylate cyclase and as an inhibitor of platelet aggregation. PGI2 and 6-keto-PGE-1 were distinguished by their halflives in citrated blood at 37°C (10 and 44 min, respectively). Fresh arterial blood contained no assayable activity after incubation for 30 min, confirming that the material measured was PGI2.

After an intravenous bolus injection of 1 nmol PGI2/kg, arterial PGI2 averaged 1.7 pmol/ml after 2 min (i.e. 90% had been removed) and returned to preinjection levels after 10 min. 6-Keto-PGE1, injected intravenously at 5nmol/kg, was cleared at a similar rate. After a bolus injection of 5 μg angiotensin II/kg, the PGI2-like material in arterial blood amounted to 7 pmol/ml after 2 min, but declined rapidly to control levels within 10-30 min. This PGI2-like material had the same half-life in vitro as authentic PGI2.

The results show that though physiologically significant PGI2 does not normally circulate in rabbits, angiotensin II can raise blood PGI2 to inhibitory levels. Formation of 6-keto-PGE1 is unlikely to contribute to the effects of PGI2 on platelets in rabbits