bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Aggregation and Plasma Catecholamines in Hypertension
Authors: Vlachakis N. D. , Aledort L.
Published: May 2019

In 19 patients, with essential hypertension (EH) while on placebo therapy and 8 age matched normals (NC), platelet aggregation studies and plasma norepinephrine (NE) and epinephrine (E) were obtained at rest and at the end of 3 min isometric handgrip exercise (IHE) (2/3 of maximal voluntary contraction) in the upright position. The percent of light transmission at 1 μM ADP concentration (LT) and the biphasic aggregation threshold (BAT) in response to ADP were measured in fresch platelet rich plasma at 300,000 platelets. In NC the LT was 26±4.8, the BAT was 2.8±0.9, NE was 242±30, pg/ml and E was 36±3.3, while in EH the corresponding values were 27.6±2.6 for LT, 3.4±1 for BAT, 222±22 for NE and 36±2.8 for E. The IHE increased LT significantly in both groups and decreased BAT significantly only in EH, while NE and E increased significantly in both groups. In 7 patients the administration of propranolol (PR) for 6 weeks or longer decreased LT and increased BAT as well as increased both NE and E, but neither change was significant. The sudden discontinuation of PR did not produce significant changes in NE, E, LT and BAT from the placebo period, when measurements were obtained one to ten days thereafter. To 7 subjects the intravenously administration of NE for 15 min produced an increase of plasma NE from 338±43 to 1774±159 associated with a significant increase in LT and significant decrease in BAT. Thus uncomplicated EH does not appear to be associated with abnormal platelet aggregation or increased NE or E. However, plasma catecholamines affect platelet function.
Prostaglandin-Endoperoxides and Cyclic 3′-5′-AMP in Platelets of Patients with Uremia
Authors: Matthias F. R. , Palinski W.
Published: May 2019

In platelets of normal donors and of patients with chronic renal failure the following determinations were performed: 1. prostaglandin-endoperoxide-formation after N-ethylmaleimide stimulation measured as malondialdehyde; 2. the c-AMP level according to the Gilman-method; 3. the adenylate-cyclase activity in response to prostaglandin E1; 4. aggregation in response to collagen.

In comparison to’normal donores the prostaglandin-endoperoxide production was reduced in uremic patients. Plasma of uremics depresses the endoperoxide formation of normal platelets. The basal c-AMP level of platelets of’ patients with renal failure was not significantly changed, whereas the plasma c-AMP level was increased; the activation of the platelets adenylate-cyclase was impaired. The adenylate-cyclase activity of platelets from normal donors was reduced by uremic plasma.. The results are of interest as to the explanation of the bleeding tendency of uremic patients.
A New Pathway for Arachidonic Acid Metabolism in Human Platelets
Authors: Rittenhouse-Simmons S. , Russell F. A. , Deykin D.
Published: May 2019

We are reporting a novel pathway of arachidonic acid metabolism in the phosphatides of thrombin-activated platelets. For kinetic studies of arachidonic acid turnover, platelet phosphatides were labeled by incubation of platelet rich plasma with (3H)-arachidonic acid for 15 min. Unincorporated isotope was removed during subsequent gel-filtration. Platelet phosphatides were resolved and quantitated following two-dimensional silica paper chromatography of chloroform/methanol extracts of incubated platelets. Plasmalogen phosphatidylethanolamine (PPE) was examined on paper chromatograms after its breakdown to lysoPPE with HgCl2. In other experiments, gel-filtered platelets were incubated with (14C)-glycerol to monitor de novo phosphatide synthesis. (3H)-Arachidonic acid was released from phosphatidylcholine and phosphatidylinositol of pre-labeled platelets exposed to thrombin and appeared increasingly in PPE in acyl linkage at glycerol-C-2. (3H)-Arachidonic acid was not found in PPE of resting cells. Maximum transfer occurred with 5 U/ml of thrombin and 15 min, of incubation, with t½ of 2½ min., and was Ca+2 dependent. The presence of aspirin, indomethacin, or eicosatetraynoic acid did not prevent the thrombin-activated transfer of (3H)-arachidonic acid to PPE. The stimulated incorporation of (3H)-arachidonic acid into PPE was not accompanied by a stimulation of (14C)-glycerol uptake into this phosphatide. We suggest that perturbation of the platelet may activate a phospholipase A2 leading to turnover of arachidonic acid in PPE, which is rich in this fatty acid. Such turnover may provide substrate for conversion by cyclo-oxygenase and lipoxydase to biologically active metabolites, and therefore, may offer a locus for regulation of prostaglandin synthesis in the human platelet.
De Novo Synthesis of Purine Nucleotides in Human Peripheral Blood Platelets
Authors: Jerushalmy Z. , Patya M. , Sperling O.
Published: May 2019

Human blood platelets were gtudied for the presence of the pathway of de novo synthesis of purine nucleotides. 8 x 108 cells were incubated for 2.5 h at 37°C in 2 ml of Eagle’s Minimal Essential Medium containing Earle’s Balanced Salt Solution, 15% fetal calf serum and 20 μCi sodium [14C] formate (59 mCi/mmole). Platelets were found to incorporate 14C into total purines at a slow but detectable rate of 50–70 pmoles/8 x 108 cells/2.5 h. This incorporation was inhibited by approximately 80% at 10 mM azaserine and by 60% at 0.1 mM adenine. Adenine is known to affect the rate of purine synthesis de novo through the activity of phosphoribosylpyrophosphate amidotransferase, the first committed enzyme of this pathway. The results suggest the presence of the complete pathway of de novo synthesis of purine nucleotides in normal human peripheral blood platelets.
The Factor VIII Molecule in Acquired Von Willebrand’s Disease
Authors: Preston F. E. , Malia R. G. , Sampson B.
Published: May 2019

Four patients with acquired von Willebrand’s disease have been studied. The diagnosis in each case was based on acquired bleeding disorder, negative family history, prolonged bleeding time, low procoagulant factor VIII (F. VIIIC) and factor VIII related antigen (F. VIIIR. A.) activity and impaired ristocetin-induced platelet aggregation responses.

Gel filtration studies were performed on plasma samples from each of the four patients and the fractions tested for F. VIIIC activity using a modified kaolin cephalin clotting time. Samples from each patient showed two peaks of procoagulant activity compared with one peak obtained on samples from controls, haemophiliacs and classical von Willebrand’s syndrome.

When incubation mixtures of acquired von Willebrand plasma and a source of normal factor VIII are examined by similar gel filtration techniques, it can be shown that the normal F. VIIIC becomes dissociated into sub-units of varying size.

Similar results have been obtained by incubating mixtures of the patients’ IgG obtained by ion exchange chromatography and normal sources of factor VIII.

It is concluded that acquired von Willebrand’s disease is probably due to an antibody directed against the factor VIII molecule.
Comparative Study of Two Procedures for Determination of Factor VIIIR:AG Levels in Normal and Hemophilic Populations
Authors: Strike L. E. , Saint-Paul B. , Allain J. P.
Published: May 2019

Factor VIIIR:AG levels were determined in three populations:37 normal males (NM), 36 hemophiliacs(H) and 36 hemophiliacs with antibodies to factor VIII (HA). The procedures used were Laurell electroimmunodiffusion (EID) and radioimmunoassay (RIA) according to Hoyer. The same rabbit antibody was used for both technics. The overall correlation between the two procedures was r=0.72. When divided according to populations, the correlations were: NM r=0.90, H r=0.72, and HA r=0.60. The clinical state of the patients was then considered, i. e., the presence of bleeding or evidence of liver disease as compared to the quiescent state. In the H population using EID, the mean VIIIR:AG level in 23 quiescent patients was 1.18±0.45 u/ml and was significantly higher in 13 non-quiescent patients, 1.52±0.28 u/ml. Using RIA, VIIIR:AG levels were 1.10+0.37 u/ml and 1.24±0.25 u/ml respectively. The correlation between the two technics was relatively high (r=0.77) for the H quiescent group and low (r=0.43) for the H-non-quiescent group. In the HA group VIIIR:AG levels in 15 quiescent patients were 1.27±0.41 u/ml with EID and 0.91+0.29 u/ml with RIA. The correlation between the technics was r=0.71. These results indicate that to properly measure VIIIR:AG levels attention should be given to the clinical state of the patient at the time of sampling, and that different technics may measure different molecular forms of the factor VIII molecule.
An International Collaborative Study of Factor VIII
Authors: Barrowcliffe T. W. , Kirkwood T. B. L.
Published: May 2019

A collaborative assay was organised to assess the suitability of a replacement for the first International Standard for Factor VIII. Coded samples of a freeze-dried concentrate (proposed 2nd I. S.), the 1st I.S., and a freeze-dried plasma were assayed by 15 laboratories against fresh normal plasma and local standards. Ten laboratories performed 1-stage assays and five 2-stage.

The proposed 2nd I. S. had a mean potency of 1.14 International Units per ampoule by direct assay against the 1st I. S.., with no significant difference between one- and two-stage assays. When assayed against a large number of individual normal plasmas, the proposed standard was equivalent to 1.05 ml “average normal plasma” per ampoule. In assays of the common freeze-dried plasma against the 1st I. S., there was a significant difference between assay methods, the 1-stage assays giving higher results for the plasma than the 2-stage. This difference between assay methods confirms results from other collaborative studies, and it seems likely that the 2-stage method is detecting relatively more activity in the concentrate standards.

It was agreed by the participants that the proposed material is suitable, in terms of stability and comparability with other materials, to serve as the 2nd International Standard for Factor VIII. The standard was established by WHO at the 28th meeting of the Expert Committee on Biological Standards, with a potency of 1.1 IU per ampoule.
Antistreptokinase Titres Before, During and after Streptokinase Therapy
Authors: Naidu R. , Kὂstering H. , Guerrero M. A. , Hampe R.
Published: May 2019

We usually prefer streptokinase therapy for thrombolytic treatment in patients with thromboembolism. For this therapy it is necessary to know the antistreptokinase (ASTK) titre, in order to calculate the initial dose, due to streptokinase resistance in some patients. The ASTK titres in 108 normal subjects and in 442 patients were examined and showed that 4.66% needed one million units streptokinase, 7.86% 500,000, 21.7% 250,000, 23.3% 100,000 and 42.5% 50,000 units. The results varied greatly in the different groups of patients.

It is sometimes necessary to repeat streptokinase administration to patients. The ASTK titre was therefore determined daily for three weeks in 25 patients before, during and after streptokinase therapy. In another group of 70 patients, who had previously received this treatment for up to 72 months, the ASTK titres were elevated and 7 days following the beginning of the new treatment had increased to 15 million units and between the 11th and 16th day to 30 million units. This was followed by a slow but steady decrease to 6 million after one month, 2.5 million after 2 months and 900,000 after 3 months. After six months were were most often normal values and low titres were rarely seen. This means that thrombolytic therapy could be repeated at this time.
Low Molecular Weight Factor VIII: A Comparison of the Electrophoretic Maps of the Normal and Hemophiliac Molecule
Authors: Rock G. A. , Cruickshank W. H.
Published: May 2019

Considerable debate exists as to the relative structure of normal and hemophiliac Factor VIII. In order to obtain some insight into the differences which exist at the molecular level we examined the various subunits found after dissociation of Factor VIII from these two populations.

Factor VIII was obtained by column chromatography of either cryoprecipitate or concentrated plasma on Sepharose CL6B. The protein eluting at the void volume was rechromatographed in buffer containing 0.25 M CaCl2. Under these conditions, both normal and hemophiliac Factor VIII underwent dissociation with an identical elution pattern. The low molecular weight faction from normal Factor VIII displayed procoagulant activity: that from the hemophiliac did not.

As determined by their elution volume and ultrafiltration, the molecular weights were identical. When digested with pepsin (protein: pepsin 50:1) for 16 hours at 37°C and “mapped” by pH 6.5/2.1 paper electrophoresis the two compounds were significantly different in the neutral and acidic peptide regions and identical in the basic region.

It therefore appears that the inactivity of hemophiliac Factor VIII is a consequence of an amino acid substitution or deletion in the low molecular weight portion of the molecule rather than a complete absence of the procoagulant molecule.
Platelet Function in Hyperbetalipoproteinemia
Authors: Colman Robert W.
Published: May 2019

Individuals with familial hyperbetalipoproteinemia are at increased risk of premature atherosclerosis and thrombosis. Although there is controversy whether platelet survival is shortened or normal in this disease, several in vitro tests of platelet function after stimulation with ADP including electrophoretic migration, platelet factor 3, aggregation response and release of nucleotides have been found to be increased. These functional changes are accompanied by an increase of cholesterol to phospholipid ratio in the platelet membrane. Clofibrate and halofenate reverse some of these abnormalities in vitro and the former drug, when administered for 6 weeks to patients with type IIa hyperlipoproteinemia decreases platelet sensitivity to ADP and epinephrine. The platelet hypersensitivity to aggregating agents can be reproduced in vitro by increasing the cholesterol to phospholipid rather in normal platelets. These artificially hypersensitive platelets can be returned to normal by halofenate in vitro. Incorporation of cholesterol into platelet membranes increases the basal level of the membrane associated enzyme adenylate cyclase. However, the enzyme no longer responds to stimulation by prostaglandin E1, and this is associated with relative resistance of the platelet to inhibition by this pharmacologic agent. These functional alterations produced by cholesterol enrichment of platelet membranes occur in parallel with an increase in platelet membrane microviscosity suggesting that the more rigid membrane can alter the behavior cf membrane associated enzymes and receptors.
Platelet Suppression Therapy in Arterial Disease: Present Status
Authors: Blakely J. A.
Published: May 2019

Arterial disease is a severe test of clinical trials methodology. Results to date have defined areas for further study, but clinical indications are not established. Dipyridamole reduces emboli from prosthetic heart valves but applicability to less thrombogenic valves is uncertain. Transient cerebral ischemic attacks are physiologically appropriate and there is preliminary evidence of reduced attacks with Sulfinpyrazone, none with Dipyridamole, and favorable case reports with Aspirin. Effects on stroke and death in patients presenting with TIA or stroke are under study. Sulfinpyrazone has failed to prolong patency time after peripheral vascular surgery. Administration of aspirin and of Sulfinpyrazone to elderly populations has shown no detectable benefit from Aspirin, and has suggested that Sulfinpyrazone should be further studied in patients recovered from thrombotic stroke. The greatest potential benefit may be secondary prevention of myocardial infarct, but to date efficacy has neither been demonstrated nor excluded. Studies in progress include: ASA and/or Sulfinpyrazone and TIA, stroke and death in patients with TIA; ASA with and without Dipyridamole in patients with TIA: Sulfinpyrazone and survival after recovery from thrombotic stroke; Secondary prevention of myocardial infarct with ASA, ASA or ASA + Dipyridamole, Sulfinpyrazone, and Clofibrate; Sulfinpyrazone, ASA, and ASA with anticoagulants and emboli from prosthetic valves. Platelet survival studies may permit selection of populations likely to benefit and assessment of adequacy of therapy.

Hypotheses tested by clinical trial must be distinguished from hypotheses formulated from retrospective analysis, and methods must permit effects of treatment to be distinguished from differences in risk.
The Role of Endothelial Cell Injury and Platelet Response in Atherogenesis
Authors: Harker L. A. , Ross R. , Glomset J.
Published: May 2019

Endothelium forms a resistant barrier between flowing blood and vessel wall structures. Endothelial thromboresistance is maintained in part by the synthesis of prostacyclin, a potent prostaglandin inhibitor of platelet function. Loss of endothelial cells, mediated by physical, chemical, infectious or immune mechanisms, exposes the sub endothelium to flowing blood. Platelets react to the subendothelial connective tissue structures, undergoing adhesion and release of intracellular constituents, including a factor that is mitogenic to smooth muscle cells. This growth factor is a heat stable, basic protein (IP 7.4–9.4) of 20,000 Daltons and appears to be responsible for the intimal proliferation of smooth muscle cells that follows endothelial cell desquamation. After a single injury event the intimal lesion regresses over several months. Repeated or continuous endothelial cell loss results in progressive intimal proliferation of smooth muscle cells, their secretion of connective tissue matrix components (collagen, elastin and proteoglycans) and accumulation of lipid when animals are on a hypercholesterolemic diet to form early atherosclerotic intimal lesions. Discontinuance of endothelial injury and restoration of the endothelium appear to be followed by lesion regression except when lipid accumulation is extensive. Possible approaches to atherosclerosis prevention include: 1) protection of the endothelium by interruption or avoidance of endothelial injury factors, and perhaps by pharmacologic protection; 2) inhibition of platelet reactivity; 3) modification of SMC proliferation, secretion or lipid accumulation.
Prevention of Arterial Thromboembolism with Acetylsalicylic Acid in Patients with Prosthetic Heart Valves
Authors: Dale J.
Published: May 2019

A double-blind study has been performed in patients with aortic ball valves in order to study the antithrombotic effects of one gm of acetyl-salicylic acid (ASA) daily combined with anticoagulants, as compared with that of anticoagulants alone. The combined treatment offered a significantly better protection, the incidence in the two groups being 1.8 and 9·3 arterial thromboembolic complications per 100 patients per year, respectively. Encouraged by these results, a pilot study on the effects of ASA alone was started in 77 patients from both groupa of the first study. They all received one gm of ASA daily, then the dose of anticoagulants was reduced gradually and the drug discontinued on average five weeks later. Six arterial thromboembolic episodes occurred in five patients during the following five months, the incidence being 14.5 complications per 100 patients per year. The study was therefore ended, and anticoagulant therapy re-instituted in addition to ASA. The results indicate that combined treatment with ASA and anticoagulants more effectively inhibits arterial thrombus formation than does each of the drugs alone.
Effect of Angiography on Blood Coagulation
Authors: Krause W. H. , Lang A.
Published: May 2019

It is known, that angiographic contrast media have an anticoagulant activity in vitro. The purpose of the present study was, to investigate this effect in vivo. The catheter was introduced percutaneously according to Seldinger into the femoral artery. The prothrombin time, activated thromboplastin time (APTT), thrombin time, reptilase time, fibrinogen, plasminogen, antithrombin III, platelets, fibrin/fibrinogen degradation products (FDP), haematocrit and contrast media concentration were studied in a series of 50 patients before and following abdominal aorto-arteriographic procedures up to 6 hours. Thrombin time and reptilase time were prolonged significantly 30 and 60 minutes after angiography. There was a correlation between clotting tiies and contrast media concentrations. Prothrombin time, APTT, and platelet counts remained unchanged. Fibrinogen, plasminogen,and antithrombin III levels showed a significant reduction after 30 minutes. FDP concentrations were increased significantly up to 6 hours, there was no correlation between contrast media concentrations and split products. The results were corrected for contrast media dilution according to the haematocrit. No thromboembolic complication was observed. The results suggest that angiographic procedure may initiate an intravascular coagulation with an activation of the fibrinolytic system. In addition the contrast media showed an inhibition of fibrin polymerization in vivo.
The Effect of the Fibrinogen Concentration and White Cell Count on Intravascular Fibrin Deposition
Authors: Gurewich Victor , Lipinski Boguslaw
Published: May 2019

The relationship between blood fibrinogen concentration and intravascular fibrin deposition was examined in EACA-treated rabbits infused with a standard amount of thrombin or simplastin sufficient to produce fibrin monomer (FM) but not defibrination. Fibrin in organs was measured by a previously described quantitative technique using 125I-fibrinogen. A significant (p<0.01) positive correlation was found between the baseline fibrinogen concentration and fibrin deposition 3 hours after infusion. By contrast, in vitro there was an inverse relationship between fibrinogen concentration and enzymatic clotting as well as non-enzymatic fibrin formation from soluble FM. When HN2-treated leukopenic rabbits were infused, fibrin deposition was inhibited despite the fact that the animals’ fibrinogen concentration was substantially increased by the HN2 treatment. When leukocytosis was induced by pretreatment with endotoxin, fibrin deposition was potentiated. It is concluded that fibrin deposition from circulating FM is facilitated by a raised fibrinogen concentration by a mechanism that cannot be explained by the well known in vitro interaction between FM and fibrinogen. Secondly, white cells appear to participate in the intravascular precipitation of circulating soluble FM complexes.
Collagen Carbohydrates are not Involved in Collagen-Initiated Platelet Aggregation
Authors: Gordon Richard K. , Simons Elizabeth R.
Published: May 2019

There is strong evidence that the quaternary (polymeric) structure of collagen is necessary for platelet aggregation. Although the specific groups required are unknown, the carbohydrate moieties have been implicated. To further elucidate their role we have used NaI04 (0.01M, 4°C in the dark for 24 hours) to selectively destroy 75–87% of total carbohydrate »The collagens were characterized by circular dichroism spectra and by polymerization kinetics observed by light scattering. The fibril formation kinetics for control collagen exhibit a linear rate for more than 5 min. while the modified collagen’s levels off to a zero rate after 1 min. Further evidence for abnormal polymerization is its inability to form fibrils in 0.25ml PPP plus 0o75ml 0.9% NaCI. However, soluble control collagen initiated normal PRP aggregation as did fibrils of control and periodated collagen preformed in buffer, although the periodated fibrils evoked a slightly attenuated response. In competition experiments, periodated monomeric collagen incubated with platelets (and thus incapable of polymerizing) delayed platelet aggregation by preformed control collagen fibrils. As concentration of modified tropocollagen increased, the lag time before platelet aggregation increased. These results suggest that preformed fibrils must displace monomeric, essentially carbohydrate-free, collagen from the platelet surface before the platelet-collagen aggregation response can occur. Thus neither the carbohydrate moiety nor the quaternary structure is necessary for collagen binding to the platelet surface. The quaternary fibrillar structure is necessary for platelet aggregation while the carbohydrates are important for the formation of such fibrils rather than for platelet aggregation itself.
Two Variants of Canine von Willebrand’s Disease
Authors: Dodda W. Jean , Benson Roger E.
Published: May 2019

Previous studies in our laboratory have characterized von Willebrand’s disease (VWD) in three families of dogs: German shepherds, miniature Schnauzers, and golden retrievers (Blood, 45, 221, 1975; Thromb. Res., 7, 383, 1975). In each family the disease follows a classical pattern of autosomal inheritance; reduced factor VIII activity, factor VIII-related antigen (FVIII-RA), platelet retention and ristocetin-induced platelet aggregation; and long bleeding times; although the severity of expression varies between the breeds. Two new families currently being studied (Doberman pinschers and Scottish terriers) deviate significantly from this expected pattern. Several severely affected dogs, presumed homozygous by virtue of having two affected parents and undetectable FVIII-RA, have factor VIII activity between 20–50% of normal. Even more unusual is that mildly affected or clinically normal close relatives of these dogs have low-normal or normal factor VIII activity but very low FVIII-RA (5–25%) . However, there are also severely affected dogs from both families with classic VWD including very low factor VIII activity (<10%>) and undetectable FVIII-RA. The same monospecific rabbit anticanine factor VIII was used to quantitate and compare the FVIII-RA levels of affected and normal members of all five dog families. The explanation for finding low antigen levels in conjunction with normal biologic activity in families where complete expression of the disease coexists remains obscure.
Prevention by Suloctidil of Spontaneous Platelet Aggregates in Rats
Authors: Saunders R. N. , Burns T. S. , Stelzer M. R.
Published: May 2019

Atherosclerosis and cerebrovascular disease have been reported by Wexler and True (Circ. Res. 12: 659,1963) to-occur in retired breeder rats but not in virgin rats of the same age. We have discovered that retired breeder (RB) male rats have spontaneous circulating platelet aggregates (CPA) as determined by the method of Wu and Hoak (Lancet 2: 924, 1974). These CPA respond to therapy with clinically proven antiplatelet drugs. Suloctidil (S) [l-(4-isopropyl-thiophenyl)-2-n-octylamino propanol, Continential Pharma, Brussels] administered for 8 days at 100 mg/kg i. g. showed a significant reduction in CPA from 0.76+0.05 (S.E.M.) to 0.92 + 0.03 (where 1.0 indicates no CPA). In these same rats, the endogenous platelet 5-HT level was significantly (P<0.01) lowered to 0.88+0.04 (S. E.M.) μg/10 9 platelets compared to control values of 1.66+0.08 μg/10 9platelets. Plasma 5-HT levels were not altered. In vitro rat platelet 5-HT uptake is inhibited 50% by S at 6.4 χ 10 -6M in platelet-rich plasma. In rat platelets with labeled 5-HT pools, 5 min incubation with S at 10 -5M resulted in significantly (P<0.01) greater release at 10, 20 and 30 sec after the addition of 2U/ml of thrombin. This suggests that S may decrease uptake as well as increase release of platelet 5-HT. The reduction of endogenous platelet 5-HT levels by S may be related to its observed platelet aggregate prevention activity.
Evaluation of the Antithrombotic Properties of Suloctidil in Patients with Substitute Heart Valves
Authors: Moriau M. , Ferrant A. , Beys C. Col-De , Hurlet A. , Chalant C. , Ponlot R. , Jaumain P. , Goenen M. , Masure R.
Published: May 2019

Isotopic platelet survival time (PST), platelet count, adhesiveness, aggregation, PF4 release and coagulation parameters were examined in 23 patients with prosthetic heart valves (6 mitral, 10 aortic and 7 mitroaortic Starr Edwards or Bjork valves) treated with or without suloctidil.

The patients were distributed in 2 comparative groups : the treated group with 11 patients with VKA (nicoumalone) and suloctidil (600 mg/day) and the control group with 12 patients with VKA alone. The biological parameters were performed before (the 10th postop. day) and 6 weeks after treatment with or without suloctidil.

Before treatment were : PST shortened, platelet adhesiveness and aggregation normal and PF4 release, factors I, VIII-C, VIII-R-Ag increased in both groups.

After treatment, PST returned to normal in the treated group, but remained unchanged or was more decreased in the control group. Platelet adhesiveness and aggregation were unchanged in both groups. PF4 release was reduced in the treated group and unchanged in the control group. Platelet count, factors I, VIII-C, VIII-R-Ag returned to the preoperative values in both groups.

Two severe thromboembolic complications appeared in the control group, none in the treated group.
A Study of Polymerization Sites in Bovine Fibrinogen
Authors: Tranqui L. , Chapel A. , Suscillon M.
Published: May 2019

Abnormalities in bovine fibrinogen polymerization have been brought about by slight proteolysis on the native molecule by two purified aminopeptidases that we have called A and B and which come from bacteria. These degraded molecules have a molecular of between 272000 and 250000. The aminopeptidase A induces the Polymerization of fibrinogen into ordered structures, the period being 8 nm, when the ionic strength (I) is between 0.1 and 0.2. Fibrinogen modified by the aminopeptidase B precipitates at 4°C (0.075 < I < 0.2)into fibre with a major period of 45 nm. The action of thrombin on these modified fibrinogen molecules at I = 0.3 gives fibres each with two minor striations, whose distribution was however different. These structures were totally different from those of fibrin fibres with its typical major period of 23 nm and its three minor striations. The analysis of the polypeptide chains, the N-terminal-acids and the amino-acids composition of these degraded fibrinogen molecules enables us to differentiate the respective rolesof the polymerization sites in the N and C terminal parts of the Act and Bβ chains.

Therefore, according to our hypothesis the fibre structures obtained from fibrinogen depend on the type of the sites which are discovered on fibrinogen by enzymatic splits and by slight conformational changes.
Purification and Partial Characterization of Rat Fibrinogen (rat-Fbg)
Authors: Nieuwenhuizen W. , van Ruijven-Vermeer Irina A.M.
Published: May 2019

Rat-Fbg was purified from rat plasma using Sepharose-1ysi ne chromatography, repeated ammonium sulphate precipitation (35% saturation) and gel chromatography on Sepharose 6B.

In order to minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding, and the collected blood was treated with Trasylol and DFP.

A preparation was obtained, which was 95% clottable and showed a single band on SDS-poly-acrylamide gel electrophoresis. Alanine was the only detectable am i no-term i na 1 amino acid.

After reduction and modification of the SH groups the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit Polyacrylamide slab-gel electrophoresis in sod i urn dodecyl sulphate. The amino acid composition of the whole Fbg and of the separated modified chains were determined. The molecular weights were 61,000, 58,000 and 51,000 for Aα, Bβ and γ-chains, respectively.

In as far as the chains are concerned, our results are in contrast with the findings of Bouma et al. (J. Biol. Chem. 250(1975) 4678), who could not discriminate between Aα- and Bβ-chains in SDS-polyacry1 am i de gel electrophoresis. Evidence will be presented that this can be due to Aa-chain degradation caused by incomplete inhibition of proteolytic enzymes during the purification.

It is concluded, that complete inhibition of proteolytic activities in all purification steps is essential to obtain native fibrinogen. Moreover, in contrast to the conclusions of Bouma rat-Fbg does not differ essentially from Fbg from other mammalian species.
Detection of Acquired Inhibitors of Factor IX with Precipitating Rabbit Antisera Against Factor IX
Authors: Ørstavik K.H. , Nilsson I.M.
Published: May 2019

The effect of acquired inhibitors of factor IX on factor IX antigen was tested in a modified electroimmunoassay with a rabbit antiserum against factor IX. Plasma from three patients with hemophilia B and inhibitor against factor IX was mixed with agarose and casted in a one cm high intermediate gel between the sample wells and the agarose gel containing the rabbit antiserum. The precipitation of factor IX antigen by the rabbit antiserum was prevented when factor IX antigen migrated through the intermediate gel containing plasma from two of the patients with inhibitor titre 7.9 and 0.8 U/ml. The concentration of inhibitor plasma necessary to reduce by 50% the amount of precipitated factor IX antigen from 10 μl normal plasma could be determined for the two patients and was approximately 2% for the patient witli titre 7.9 and 15% for the patient with titre 0.8. The third patient had a titre of 0.13 U/ml and only a slight reduction in precipitated antigen was obtained with a high concentration of plasma in the intermediate gel (30%). The precipitation of factor IX antigen from 7 patients with hemophilia B+ (factor IX antigen 17-109%)and one patient with hemophilia BM (factor IX antigen 98%) was also inhibited by plasma from the two patients with inhibitor titre 7.9 and 0.8. This study suggests that absorption in intermediate gel may be a simple technique for the detection of inhibitors with titre 0.8 and higher and also for the study of the specificity of inhibitors.
Heparin Tolerance Determinating Factors Evaluated by the Heparin Thrombin Clotting Time
Authors: Andersen P. , Godal H.C.
Published: May 2019

The anticoagulant effect of heparin in test systems composed of purified antithrombin III, fibrinogen and thrombin, proved by far more sensitive to heparin than was whole plasma, indicating that components of platelet poor plasma (besides antithrombin III and fibrinogen) strongly interfere with the anticoagulant effect of heparin. Among the so-called acute phase proteins, orosomucoid and alpha1-antitrypsin were especially active, probably acting as competitive inhibitors of the heparin-antithrombin III formation. Thus, inhibition of heparin by orosomucoid (final concentration 1 mg/ml) was stronger than the heparin-neutralizing effect of crude platelet factor 4(PF-4) obtained by freezing and thawing of a platelet suspension containing 200 × 103 platelets/ul. Since orosomucoid and alpha1-antitrypsin are both markedly increased in various clinical situations associated with increased frequency of thrombosis, these glucoproteins may prove important contributors to the increased heparin tolerance observed under these circumstances.
Comparison of in Vivo Biochemical Effects of Human Urokinase Prepared from Urinary and Tissue Culture Sources
Authors: Marder Victor J. , Donahoe Joseph F. , Bell William R. , Cranley John J. , Kwaan Hau C. , Sasahara Arthur A. , Barlow Grant H.
Published: May 2019

In patients with pulmonary emboli, resolution has been shown to be more rapid in those receiving urinary urokinase than in those receiving heparin alone. A different source of urokinase has now been developed, namely human kidney cells grown in tissue culture. In a randomized, multicenter trial, two groups of 15 patients with pulmonary embolism received either the urinary or tissue culture urokinase. Blood samples prior to, during and after treatment were compared with regard to biochemical changes in the plasma fibrinolytic system. Both agents caused strikingly similar rates, degrees and durations of response, as reflected in the whole blood euglobulin lysis time, unheated fibrin plate lysis zones, 125-I tagged clot lysis, plasma plasminogen, plasma clottable protein and serum fibrin/fibrinogen degradation products. Bleeding occurred in about 50% of both groups of patients, primarily from cutdown sites. The results clearly indicate that the pharmacologic effect of tissue culture urokinase was the same as that of urinary urokinase, and it is reasonable to expect that both materials will be equally effective in the hemodynamic and clinical aspects of patients with pulmonary embolism.
Studies on Platelet Glycoproteins by Surface Labeling and SDS Polyacrylamide Gel Electrophoresis
Authors: Hagen I. , Solum N.O. , Peterka M.
Published: May 2019

Platelet surface (glyco)proteins, and alterations in these in connection with the thrombin-induced release reaction has been studied. Platelets were labeled by lactoperoxidase-catalyzed iodination, and examined by SDS gel electrophoresis in two different gel systems, one conventional(J. Biol. Chem.1969 244 4406), and the other containing urea and EDTA in the gels. In the conventional system the bulk of radioactivity coincided with a PAS band (GP III) of MW about 100, 000. In the other system, the main radioactive peak appeared in the GP II area (MW 120,000), and a shift in the PAS stain intensity from GP III to GP II was seen. Thrombasthenic platelets showed only traces of the GP II band in both systems. The bulk of radioactivity was associated with the surface glycopolypeptide GPS, which is present, but not labeled in normal platelets. In thrombin-released platelets, GPS in its unreduced state has an altered electrophoretic mobility compared to control platelets and platelets which have been incubated with metabolic inhibitors to prevent secretion. The findings indicate that the GP III band consists of two different polypeptides, one of which appears in the GP II area in gels containing urea and EDTA. Further, that thrombasthenic platelet membrane exists in a conformational state different from that of normal platelets. And finally, GPS is in some way involved in, or influenced by, the thrombin-induced release reaction.
Further Studies on the Glycoprotein Composition of Normal Human, Bernard-Soulier and Thrombasthenic Platelets
Authors: Nurden A.T. , Caen J.P.
Published: May 2019

The glycoprotein composition of normal human platelets has been more precisely defined using SDS-polyacrylamide gel electrophoresis and comparing SDS-phosphate, SDS-tris and SDS-trisglycine buffer systems. At least 10 glycoproteins may be identified including a previously unlocated high M. Wt. (in excess of 500,000 daltons) membrane glycoconjugate, glycocalicin and the intracellular thrombin sensitive protein. A much diminished staining for carbohydrate of membrane GP la and lb, and of glycocalicin, were the major characteristics observed for Bernard-Soulier (B-S) platelets which also gave a reduced agglutination with wheat germ lectin. These abnormalities were much greater than the changes observed using neuraminidase-treated control platelets. The B-S platelets aggregated rapidly in the presence of thrombin, an event difficult to reconcile with glycocalicin being the proposed receptor for thrombin on the platelet surface. Membrane GP lib and also GP Ilia (depending on the conditions of membrane solubilization and the subsequent SDS-polyacrylamide gel elctrophoresis) both appeared to be absent in thrombasthenic platelet membranes. These abnormalities were often accompanied by an increased staining for carbohydrate of the members of the GP I family. With both the control and the pathological platelets the rate of migration and the staining intensities of the glycoprotein bands depended on the gel buffer system and the presence or absence of reducing agent during the initial soubilization. The results have indicated structural relationships and possible interconvertions between individual GP bands. They have also more closely defined the glycoprotein abnormalities in B-S and thrombasthenic platelets.
Ultrastructural Study on in Vivo Blood Compatibility of Biolized Materials
Authors: Harasaki H. , Picha G. , Helmus M. , Fields A. , Kiraly R. , Nose Y.
Published: May 2019

The long-term implantation of cardiac prostheses has been hampered by the lack of thromboresistant blood interfacing materials. Glutaraldehyde treated pericardium and glutaraldehyde treated, gelatin-coated rubber have been utilized for the blood contacting materials of our devices. These “biolized” cardiac prostheses were implanted in calves: 8 total artificial hearts for up to 5 months, 7 left ventricular assist devices for up to 7 months, and a passive pump implanted in the aorta for 5 1/2 years. The blood-solid interaction and endothelialization of these materials were chronologically followed at an ultrastructural level using scanning and transmission electron microscopy.

The devices implanted up to 40 days were covered by a thin layer of fibrinous material. The adhesion of blood corpuscles were minimal except for the region of diaphragm-housing junction. The majority of the adhered platelets were “contact” and “spread” platelets. Once this fibrin-rich layer was formed, no significant thrombus formation was observed. The endothelialization was observed in the portion of the pericardium surface adjacent to the inflow valve in the heart implanted for 145 days. The coverage of the endothelial cells was more complete in the passive pump. Although Weibel-Palade bodies were observed in the cytoplasm of these cells, they were characterized by the sparse microvillous projections, small number of pinocytotic vesicles, and widely opened intercellular junctions. In the subendothelial layer, no basement membrane was observed.

The study showed the aldehyde treated collagen and gelatin are suitable materials as blood contact surfaces of the cardiovascular devices.
Radioimmunoassay of Human Platelet Factor 4
Authors: Chesterman C.N. , McGready J.R. , Morgan F.J.
Published: May 2019

Plasma levels of the platelet specific protein, platelet factor 4 (PF4), may reflect alteration in platelet turnover or activation. Measurements more sensitive and specific than bioassay are required to test this hypothesis.

Highly purified human PF4 prepared by affinity chromatography on heparin/agarose and cation exchange chromatography has been used to develop a sensitive radioimmunoassay. A standard double antibody technique with goat antirabbit serum as second antibody has been used. Anti-PF4 antibody was raised in rabbits with the multiple subcutaneous injection technique. Following a single booster an antibody was obtained which, at a final dilution of 1:75,000, binds 37% of tracer PF4. Iodinated tracer PF4 was produced by the lactoperoxidase labelling technique and purified by gel chromatography in heparin containing buffer to prevent absorptive losses. The assay buffer also contains heparin 1 U/ml.

The tracer shows good stability (4-8 weeks) and adequate immunoreactivity. Non-specific binding was 2.4 ± 0.8% in 11 consecutive assays. Sensitivity of the assay, expressed as 50% displacement of the tracer, is normally 0.3-0.4 ng per tube with a working range of 0.06-1.25 ng (20-80% displacement). Twelve replicate determinations of a single plasma sample yielded a within-assay standard deviation of 0.6 ng/ml and coefficient of variation of 0.1%. The assay is highly specific, the closest cross reactivity being obtained with β-thromboglobulin which produces 50% displacement of tracer with 5 μg/tube. Plasma preparation may not yet be optimal, but normal values in carefully prepared samples lie below 10 ng/ml.
Detection, Purification and Comparison of Factor IX-Like Molecules
Authors: Bertina R.M. , Veltkamp J.J.
Published: May 2019

An antibody specifically precipitating human factor IX has been raised in rabbits using highly purified factor IX (batchwise DEAE-Sephadex adsorption and elution, chromatography on heparin-Sepharose, disc-polyacrylamid gel electrophoresis; MW(SDS), 67,000).

With an electro-immuno-assay for factor IX 6 hemophilia B+ families were detected (only one of which showed a prolonged prothrombin time with ox-brain thromboplastin). All factor IX like material demonstrated normal electrophoretic mobility (when mixed with normal plasma) under standard conditions.

From one patient (hemophilia BM) the factor IX-like material has been purified at least 3000 times and compared with normal factor IX. The factor IX-like material from all B+ families has been compared with normal factor IX with respect to their behaviour in 2-dimensional electrophoresis at different time intervals after the initiation of contact-induced coagulation. The precipitation peak of normal factor IX gradually disappears after activation. Improvement of the precipitation conditions during electrophoresis by the addition of poly ethylene glycol at least partially prevents this phenomenon.

Finally, it is concluded that the use of a precipitating antibody against factor IX is a valuable tool in the study of factor IX-like molecules.
Activation of Human Factor X
Authors: Orthner Carolyn L. , Morris Sam , Kosow David P.
Published: May 2019

Factor X is the zymogen of the proteolytic coagulation enzyme Factor Xa. Since the activation of Factor X to Factor Xa may be a rate limiting step of the coagulation cascade we have begun investigations of the mechanism of this reaction. Human Factor X has been purified 6000-fold from human plasma and the final product is over 95% pure as judged by Polyacrylamide gel electrophoresis. Human Factor X has a monomeric molecular weight of 75,000 and consists of two chains held together by a disulphide bridge. The molecular weight of the heavy chain is 56,000 and that of the light chain is 17,500. The venom coagulant protein of V. russelli (RVV-X) activates human Factor X by cleaving the heavy chain. When fully activated, human Factor Xa shows two bands on Polyacrylamide gel electrophoresis indicating that human Factor Xa like the bovine enzyme has two molecular forms.

The kinetic mechanism of the activation reaction has been investigated utilizing the chromogenic Factor Xa substrate Bz-Ile-Glu-Gly-Arg-p-Nitroanilide (S-2222). The reaction has an absolute requirement for Ca; Mg cannot substitute for Ca, however Mg can increase the Vmax of Xa formation in the presence of suboptimal concentrations of Ca. Both Ca and Mg effects exhibit positive cooperativity. Our data indicate that human Factor X has at least three cooperative metal binding sites some of which are specific for Ca.
A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom
Authors: Markland F.S. , Chou J. , Shih Y. , Pirkle H.
Published: May 2019

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.