bluuebunny/crossref_metadata_2025 huggingface dataset:
Vein Contraction And Smooth Muscle Cell Extensions As Causes Of Endothelial Sloughing During Graft Preparation
Authors: Baumann F G , Catinella F P , Cunningham J N , Spencer F C
Published: July 2018

In this study light, transmission and scanning electron microscopy were used to investigate the effects of various methods of vein graft preparation on endothelial and smooth muscle cells (SMC) of the dog cephalic vein. After removal, veins were stored in one of three heparinized solutions at 10°C for 5 minutes or 1 hour: autologous blood, Plasma-Lyte or Plasma-Lyte with 0.6 mg/ml Papaverine added. The vein wall proved very sensitive to dissection, manipulation and introduction of fixative and reacted to such stimuli with a severe contraction which not only diminished the luminal diameter, but also resulted in formation of medial SMC cytoplasmic extensions and protrusion of the endothelial cells into the lumen. The SMC cytoplasmic extensions were particularly frequent in the immediate subendothelial area and seem to play a role in lifting up, separating or desquamating the endothelial cells. Such findings also are relevant to the chronic effects of arterial spasm. Among the stored veins, those soaked in blood showed the greatest vessel wall contraction and endothelial cell loss. Veins soaked in Plasma-Lyte-Papaverine had the most relaxed and normal appearance and the least endothelial cell loss. If the Papaverine treated veins were subsequently subjected to brief distension at 100 mmHg, however, large gaps appeared between the endothelial lining cells. The results suggest that Papaverine treatment greatly reduces vein graft endothelial cell loss due to contraction, but Papaverine-induced relaxation should be reversed before the graft is subjected to arterial pressure.
Effect Of Removing Sialic Acid From Vascular Endothelium In Living Animals On Its Interaction With Circulating Platelets
Authors: Born G G V , Görög P , Schraufstätter Ingrid
Published: July 2018

The non-adherence of platelets in the circulation has been attributed mainly to two mechanisms : electrostatic repulsion due to net excess negative surface changes on platelets and endothelial cells; and release of endothelial prostaglandin I2 (prostacyclin) which inhibits platelets. CUr experiments were intended to provide evidence on the relative contribution of these mechanisms.

Rabbits were injected with 3H-S-hydroxytryptamine to label the circulating platelets. After about 2h, the rabbits were anaesthetised with nembutal. Both common carotid arteries were constricted in two places about 3 cm apart and perfused free of blood. One common carotid was perfused with saline as control and the other with neuraminidase (from Signa or Behring Werke : 0.5 u/ml saline) for 30 min. These conditions apparently brought about maximal release of sialic acid from the endothelial surface. The constrictions were removed from the arteries to restore the blood flow. After 10 min both carotids were removed. Separate segments were prepared for determinations of radioactivity and prostaglandin I2 and for microscopy.

In seven experiments, the effects of neuraminidase was to increase the deposition of platelets, as measured by their radioactivity, about 4-fold (mean : range 2.0 - 7.3) above controls. Neuraminidase had no significant effect on prostaglandin I2 production, determined by bioassay and radioimmunoassay.

It is concluded that diminution in electrostatic repulsion between circulating platelets and vascular endothelium by removal of sialic acids from, endothelial surface glycoproteins permits adherence of platelets irrespective of prostaglandin I2.
Spontaneous Platelet Aggregation In A Hereditary ‘giant’ Platelet Syndrome (MPS)
Authors: Milton J G , Tang S S , Frojmovic M M
Published: July 2018

Spontaneous platelet aggregation (SA), induced by stirring citrated platelet-rich plasma (PRP), has been observed in a large number of patients with arterial insufficiency and shown to be mediated by plasma and/or platelet-released factors. Here we examine the characterisitcs of SA in a hereditary giant platelet syndrome, the Montreal Platelet Syndrome (MPS), and demonstrate a distinction from that observed for patients with arterial insufficiency. SA was quantitated by microscopy from the decrease in single platelet concentration in PRP. In contrast to normal PRP, a significant proportion (20-50%) of platelets in MPS-PRP occurred in micro-aggregates typically containing 2-6 discshaped platelets. Stirring MPS-PRP at 1000 rpm for 10 minutes further increased the fraction of platelets in aggregates by 10-170%: the % increase not being correlated to the donor's platelet count (5,000 - 220,000 μl-1). Normal platelets re-suspended in MPS platelet-free plasma (PFP) did not undergo SA, whereas MPS platelets re-suspended into normal PFP or calcium-poor Tyrode's continued to show SA. The increase in SA upon stirring was only partially inhibited by 1 μM PGEl (69-85% inhibition), 1 μM C1-adenosine (26-43%) and 0.3 mg/ml apyrase (18-25%), i.e. at concentrations equal to or greater than that necessary to completely inhibit SA in PRP from donors with arterial insufficiency. SA in ACD-PRP was much less than in PRP (e.g. 6% versus 30%); however, SA re-occurred on returning the pH to 7.4. SA was observed for 5/5 MPS donors, but only 1/3 of these donors had platelets with both reduced/altered glycoprotein I and reduced sialic acid while the other 2 siblings' platelets showed no such abnormalities. This indicates that neither of these entities are directly related to the etiology of SA in MPS. The above observations distinguish SA in MPS from that reported for arterial insufficiency and point to the existence of an as yet undetermined anomaly of the plasma membrane as the basis of the etiology of spontaneous aggregation in MPS.
Congenital Thrombocytopenia With Giant Platelets: An Ultrastructural Morphometric Study
Authors: Barnhart M I , Ragab A H , Lui V , Evatt B L , Lusher J M
Published: July 2018

We have studied a family with a previously undescribed familial platelet abnormality. 5 year old twins were evaluated because of excessive bruising and bleeding since infancy. Their mother also had a moderate bleeding tendency. All three had slightly decreased numbers of platelets (P) of variable size, abnormally low prothrombin consumption (which corrected with a P substitute), and abnormal P factor 3 availability (which corrected with disruption of P by repeated freezing and thawing). P aggregation was normal with ADP, epinephrine and ristocetin, but absent upon addition of collagen. SEM revealed abnormal surface activation upon contact, with the multi-podded giant P showing little evidence of spreading capability. In all 3 subjects P morphometry confirmed the impression of giant P. Whereas only 10% of normal control P had a maximum diameter (D max) of >3um, 77-82% of the twinsβ P had a D max of >3um, while 40% of the mother’s P did as well. Whereas only 5% of normal P had a surface area (SA) of>4.5 um2 , 55 and 93% of the twins' P and 50% of their mother’s P had>4.5 um2 SA. Morphologically a number of P profiles had extensively vacuolated interiors (twins had 5-6 X normal). Other P sections contained only a few organelles. We applied quantitative morphometry to P sections. When organelles were counted per um2 of P, all 3 family members had mitochondria and ∝ granules in the normal range, whereas all 3 had a slight reduction in dense bodies. (These findings rule out a diagnosis of gray platelet syndrome). The ∝ granule compartment, although of normal numbers, displayed a higher than normal proportion of mixed type granules (dense component, bulls eye configurations in a lower density homogeneous matrix). Multiple defects (20% or more vacuolated P, reduced dense bodies, abnormal surface membrane) appear to be responsible for ineffective platelet plug formation.
Analysis Of Chemical Heterogeneity And Purity Of Commercial Heparins
Authors: Hurst R , Lurie A , Settine J , Corum M
Published: July 2018

The nature of chemical heterogeneity in commercial heparin was investigated, and a practical method of characterization and assay of pharmaceutical heparins was developed. One beef-lung (BL) and six hog-mucosal (HM) heparins were fractionated by sequential extraction of their hexadecyl- pyridinium complexes in 1-butanol by aqueous solutions containing successively increasing concentrations of NaCl. This system fractionates heparins larger than 10,000 daltons according to their anionic density. The fractions were characterized by molar ratios of carbohydrate constituents and by their anticoagulant potencies in the APTT test with human plasma. Galactosamine content and the amount of uronate extracted with less than 0.14 M NaCl provided a measure of non-heparin GAG and low-quality heparin. The molar ratios of carbohydrate constituents of equivalent fractions from different HM heparins were similar, and with the exception of one unbleached sample, had equivalent anticoagulant potencies which were linearly related to the square of the anionic density. The BL-heparin was clearly distinguishable from the HM-heparins in that its most abundant fractions were extracted at higher NaCl concentrations, and the systematic variation in anionic density seen with HM- heparins was not evident. The fractions were only about half as potent as the equivalent HM-heparin fractions.

A simplified version of the extraction with a two-step partition fractionation was used to measure the percentages of non-heparin GAG and high-quality heparin respectively, and measurements of various molar ratios were used to both characterize the chemical properties of the heparin and its purity. Activation with silica in the APTT test yielded rectilinear semi logarithmic dilution plots whereas ellagic activation yielded curvilinear plots with lower sensitivity.
Acute Phase Stimulation Of Fibrinogen Synthesis. Evidence Against A Major Mediator Role For Granulocytes
Authors: Kernoff L M , Colman J , Rawlings E
Published: July 2018

This study evaluates the neutrophil granulocyte as a possible mediator of the acute phase protein response. 300g male rats received 1 ml mineral turpentine IM to create a reproducible inflammatory lesion. Five, 16 and 40 hr later i) “in vivo” plasma fibrinogen, transferrin and albumin concentrations were determined, and ii) livers were removed perfused with rat blood and the synthesis rates (SR) of these proteins measured directly by the 14C carbonate method of McFarlane and Reeve. The results in Table I demonstrate i) that both fibrinogen and transferrin are positive reactants but that the latter’s response is by comparison slower, ii) that the “in vivo” fall in albumin concentration results from increased catabolism/redistribution since the SR remains normal. In further experiments rats were rendered neutropenic (0.3mg/kg N. mustard) prior to turpentine injection and the 40 hr measurements repeated. Neutropenia, 0.26 × 109/1 (cont. = 2.17) failed to prevent the previously observed changes in protein concentration and SR (Table II). Rats receiving N. mustard only (neutropenic controls) did not differ from normal.
Isolation, Characterization, And Steroid Hormone Regulation Of Synthesis Of Fibrinogen From Xenopus Laevis
Authors: Wangh L J , Holland L J , Weisel J W
Published: July 2018

Fibrinogen was prepared from Xenopus plasma by a modified mammalian procedure. The purified protein was 95% clottable upon addition of bovine thrombin and SDS-PAGE showed four polypeptide bands which comigrated with polypeptides found in unfractioned plasma. The four polypeptides have molecular weights of 60K, 55K, 52K and 50K Daltons. Upon thrombin cleavage the 60K polypeptide yields a 53K product while the 55K and 52K polypeptides yield a product of 51K. Comparison of the cleavage products of human, bovine, and frog fibrinogens with the chain specific proteolytic venoms, copperhead Venzyme and botroxobin, demonstrates that the 60K polypeptide of Xenopus fibrinogen is the Bg subunit while the 55K and 52K polypeptides are both Aa chains. The 50K polypeptide is the γ chain. In contrast to other species, the frog Bβ and β chains are larger than the Aa and α chain. Primary cultures of purified Xenopus liver parenchymal cells maintained for several weeks in a defined culture medium secrete fibrinogen. 35s-methionine labeling of the secreted proteins showed radioactive polypeptides which comigrate with the 60K, 55K and 50K polypeptides of fibrinogen. The liver-synthesized polypeptides are selectively precipitated by a rabbit antiserum prepared against frog fibrinogen. The radioactive Aα and Bβ chains are cleaved by thrombin. We conclude that the Aα polypeptide is secreted as a 55K product but undergoes modification in circulation to give the 52K polypeptide. Synthesis and secretion of fibrinogen as well as several other proteins in liver cultures is dependent upon the continuous presence of a glucocorticoid. Wheat germ in vitro translation products of mRNA from livers of dexamethasone-treated frogs include three polypeptides of molecular weights similar to those of liver secreted fibrinogen. Thrombin selectively cleaved the two larger translation products identifying them as the frog Bβ and Aα chains. Thus, the polypeptides of frog fibrinogens are translated from separate mRNAs.
Detection And Regulation Of The Subunit Proteins Of Plasma Factor XIII
Authors: McDonagh J , Skrzynia C , Ikematsu S
Published: July 2018

Plasma factor XIII circulates as a noncovalently-associated, tetrameric zymogen (a2b2). The a subunit has the potential catalytic function, while the b subunit may act as a carrier protein plasma. In order to study the interactions between the two subunits, specific radioimmunoassays have been developed for each subunit. The assays are valid for measuring a and b protein concentrations in plasma and serum as well as in purified systems. The a assay detects all forms of the protein (zymogen, intermediate, enzyme). Factor XIII activity was measured by the monodansylcadaverine assay. These methods were used to correlate a and b protein concentrations with factor XIII activity in normal donors, in patients with factor XIII deficiency and their family members, and in patients with factor XIII inhibitors.

The normal plasma concentration of each of the subunits of factor XIII is about 12 μg/ml, making the concentration of the zymogen complex to be 0.15 μM. All of the b protein is recovered in serum, while a variable amount of a protein (a*) is found in serum. a protein and factor XIII activity go in parallel in normal, factor XIII deficient, and heterozygous plasma samples. Deficient patients have <1% activity and <20 ng/ml a protein. Deficient patients have about 50% of the normal plasma b concentration, and heterozygotes have 50% a and 75% b protein. In three cases of spontaneous, autoimmune inhibitors against factor XIII, there was no detectable activity and b concentration was one-half normal. Following transfusion of two factor XIII deficient patients with a subunit, activity rose immediately to the expected levels, while b rose more slowly to 20% above the preinfusion level. All of these studies indicate that the circulating level of functional a subunit exerts a positive feedback effect on the concentration of b subunit in plasma.
Thrombocytosis And Platelet Activation In Alcoholics During Alcohol Withdrawal
Authors: Hutton R A , Fink R , Wilson D T , Marjot D H
Published: July 2018

In order to further investigate the basis of the increased thrombotic risk reported in association with acute alcohol withdrawal, we have carried out platelet studies in a group of alcoholics admitted to a formal alcohol detoxification programme.

All patients were alcoholaemic on admission but had taken no other platelet inhibiting medications for at least two weeks. Platelet counts, platelet aggregabi1ity, plasma betathromboglobulin (BTG) assays,platelet nucleotide levels and circulating platelet aggregates were measured sequentially over a 4-6 week period.

On admission, platelet aggregabi1ity was impaired, compared to normal controls, particularly using ADP or adrenaline as agonists, but all other platelet studies were within normal limits.

Compared to normal subjects, the treated group showed a statistically significant mean increase in platelet count, the level of circulating platelet aggregates, the plasma BTG level and in the aggregation response to ADP and adrenaline. There was also a small increase in the aggregation observed with collagen and Ristocetin and in the platelet nucleotide levels but these did not reach conventional statistical significance.

The changes were not associated with any clinically overt thrombotic complications during the period of study,but may contribute to the increased incidence of thrombosis reported by others.
Platelet Survival In Relation To Vessel Wall Injury
Authors: Kinlough-Rathbone R L , Packham M A , Mustard J F
Published: July 2018

Shortened platelet survival is associated with the thromboembolic complications of vascular disease. Shortened platelet survival occurs in animals with repeated mechanical injury to the vessel wall, hypercholesterolemia, or during infusion of homocysteine; in these circumstances endothelial cells are damaged and platelets interact with the injured vessels. In rabbits, de-endothelialization of aortae with a balloon catheter does not shorten platelet survival and the injured vessel wall rapidly loses its ability to attract significant numbers of circulating platelets. In contrast, repeated mechanical injury of rabbit aortae induced by indwelling aortic catheters shortens platelet survival and platelets continue to interact with the vessel wall. When short aortic catheters are used in rabbits, thrombi continue to form but platelet survival is unaffected, possibly because damage to the vessel wall is less extensive. Mechanical injury to rat aortae shortens platelet survival without macroscopic thrombi. Thus, shortened platelet survival may occur because of increased platelet consumption resulting from platelet interaction with repeatedly injured vessel walls, or because alterations of platelets that interact with injured vessels or participate in thrombosis lead to their clearance from the circulation. Of the stimuli known to be involved in thrombosis, only plasmin, which cleaves membrane glycopeptides, alters platelets sufficiently for platelet survival to be shortened. Agents that prolong platelet survival may exert their effects by modifying the extent of vessel wall injury, modifying or inhibiting changes in platelet glycoproteins or by inhibiting the clearance of altered platelets from the circulation.
Role Of Lung Scanning In Diagnosis Of Pulmonary Embolism
Authors: Alderson P O
Published: July 2018

There is general agreement that a normal perfusion lung scan essentially excludes the presence of pathologic pulmonary emboli (PE) and that the perfusion scan pattern helps guide subsequent pulmonary angiography. The perfusion scan alone is too nonspecific to aid evaluation of patients with suspected PE in other ways but combined ventilation-perfusion (V-P) imaging is often helpful. V-P imaging does not allow a definitive diagnosis of PE, but allows the probability of PE to be determined. If normal ventilation is present in two or more regions which demonstrate subsegmental, segmental or lobar perfusion defects, there is approximately a 90% chance that emboli are present. This is true even when a patient demonstrates a mixed pattern of V-P match and mismatch or when pulmonary infiltrates or other opacities are visible on the chest radiograph in other parts of the lung. If ventilation abnormalities are present in 50% or less of the lung fields and all perfusion defects are matched by ventilation abnormalities there is less than a 5% chance for PE.

These data are derived from a recent study of 83 patients with suspected PE who underwent angiography and had V-P studies showing ventilation abnormalities and at least one area of V-P match. Angiography should thus be used selectively to evaluate patients with nondiagnostic lung scans (these account for 15-30% of most peopled experience), and to achieve definitive diagnoses in patients who are at special risk for anticoagulant therapy or who are to undergo surgical treatment of thromboembolism. V-P lung imaging is a safe, easily tolerated, low radiation dose exam which provides substantially greater confidence in the pre-angiographic diagnosis of PE than a standard clinical evaluation. For these reasons, V-P lung scanning can make significant contributions to management decisions in patients with suspected PE and alleviate the need for angiography in many instances.
Interaction Of Fibrin And Tissue Plasminogen Activator
Authors: Wallén P , Rånby M
Published: July 2018

Fibrin itself has a marked influence on fibrinolysis induced by tissue plasminogen activator (TA) indicating a specific interaction. The interaction between fibrin and TA is manifested in two ways, 1) a marked stimulating effect of fibrin on the activation of plasminogen; 2) physical ad- sorbtion of TA on fibrin. By measurement in a sensitive analytical system in which the generation of plasmin is followed by a chromogenic substrate it has been shown that TA is a rather poor activator of plasminogen. In the presence of fibrin the kinetics of the activation is dramatically changed. A stimulation up to 1000-fold is obtained at low plasminogen concentrations. As for activation of native plasminogen (Glu-plasminogen) there is a decrease of km (about 15-fold) as well as an increase of kc (about 80-fold). Fibrinogen has comparatively little effect on the activation rate (at the most 10-fold). The amidolytic activity of TA, using an activator sensitive substrate, is not influenced by fibrin indicating that the effect is not due to conformational changes in the active site region of TA.

By varying the concentration of fibrin in the test system it has been demonstrated that the stimulation effect increases suddenly at a fibrin concentration of about 0.01μM. It was suggested that this value represents the dissociation constant of the TA-fibrin complex. However, the amount of fibrin necessary for the adsorbtion of TA in purification experiments indicates a significantly higher dissociation constant (about 0.4 μM). An important difference between the purification experiments and the studies on fibrin stimulation is that plasminogen (plasmin) is absent in the former studies. The formation of a triple complex between fibrin, plasminogen and TA may be the explanation for a more efficient binding of TA in the kinetic studies.
Recent Advances In The Study Op The Mechanism Op Fierin Polymerization
Authors: Olexa Stephanie A
Published: July 2018

Fibrinogen is converted to fibrin monomer by thrombin, which cleaves the fibrinopeptides A and E. The resulting fibrin monomers spontaneously polymerize to form a fibrin network, which is later stabilized with covalent crosslinking bonds. Although the action of thrombin and Factor XHIa have been fairly well defined, the mechanism of fibrin polymerization is not yet understood. Electron microscopy and light scattering studies have provided information on the arrangement of molecules within the fiber. Monomers appear to align with a half-staggered overlap, resulting in a fiber with a width equal to twice that of fibrinogen. Detailed studies on the location and interaction of polymerization sites have been done primarily by using fibrinogen and fibrin degradation products. The studies indicate that polymerization is due to the interaction of complementary binding sites which are located on the Fragment D and the NH2- terminal domains in the fibrinogen molecule. One of these sites, located in the sequence 373410 of the gamma chain, is available on fibrinogen in the Fragment D domain. This site is complementary to the site revealed by the loss of fibrinopeptide A, which is probably located in the sequence contiguous to PPA. A second polymerization site in the NH2- terminal domain of fibrin may be located in the sequence following fibrinopeptide B. The recriprocal site may also be located on the Fragment D region. The polymerization of fibrin appears to be due to a complex interaction between molecules with both the primary sequence and the three-dimensional conformation having vital roles.
Thrombin Binding To Factor VIII/Von Willebrand Factor Protein
Authors: Switzer M E P , McKee P A
Published: July 2018

Thrombin (IIa) both activates and inactivates the procoagulant activity of Factor VIII/von Willebrand Factor (FVIII/vWF). The level of activation increases as the IIa: FVIII/vWF ratio approaches 1:1, suggesting that IIa might bind stoichiometrically to FVIII/vWF either during or after activation. We approached this question by gel filtration and ultracentrifugation studies of FVIII/vWF and l25I-IIa, which activated FVIII/vWF as well as unlabeled IIa. When the mixture of 125I-IIa and FVIII/vWF was chromatographed on 4% agarose a peak of 125I-IIa was eluted with the FVIII/ vWF in the void volume (V0). Similarly, when 125I-IIa was ultracentrifuged with FVIII/vWF, a peak of radioactivity sedimented with the FVIII/vWF protein. 125I-aibumin, used to approximate a control, did not bind to FVIII/vWF. The 125I-IIa-FVIII/vWF complex isolated from the 4% agarose filtration retained ∼50% peptidase activity. The ability to activate additional FVIII/vWF or to clot fibrinogen was <10% of that of free IIa isolated from the same chromatogram. Both the FVIII and vWF moieties appear to be important in binding, since VD protein isolated from the gel filtration of FVIII/vWF on 4% agarose in 0.25 M CaCl2 binds about 24% as much 125I-IIa as native FVIII/vWF. When the isolated 125I-IIa-FVIII/vWF complex was rechromatographed on 4% agarose in 0.15 M NaCl, essentially no dissociation occurred. When these experiments were repeated in 4 M guanidine hydrochloride (GnHCl), ∼30% of the IIa remained bound. When the 125I-IIa-FVIII/vWF complex was isolated from the GnHCl chromatography and analyzed by SDS-PAGE, 58% of the IIa remained bound to the FVIII/vWF before reduction and 43% of the IIa remained bound even after reduction with β-mercaptoethanol for 3 hours at 37°. Thus FVIII/vWF binds at least some of the IIa very tightly. Since FVIII/vWF-bound thrombin is essentially inactive toward macromolecular substrate, binding of thrombin to FVIII/vWF is most likely a mechanism for removing active thrombin from the circulation.
The Effect Of Coagulation On The Association Of VIII:CAg With VIIIR:Ag
Authors: van Mourik J A , Lantinga P H G , Hellings J A
Published: July 2018

Solid-phase immunoradiometric assays specific for Factor VIII coagulant antigen (VIII:CAg) and Factor VIII related antigen (VIIIR:Ag) have been used to assay the immunoreactivity of these antigens in plasma and whole blood during coagulation at physiological calcium concentration. When non-anticoagulated plasma, prepared from blood immediately after venipuncture, was incubated at 37°C, the concentration of VIII:CAg and VIlIR:Ag did not change. However, when whole blood, collected withoutanticoagulant, was incubated, the concentration of VIII:CAg gradually decreased to 50% of the initial value whereas the concentration of VIIIR:Ag remained unchanged. Gelchromatographic analyses revealed that coagulation of plasma leads to progressive dissociation of VIII:CAg from the factor VIII:VWF complex. When plasma was chromatographed before the onset of coagulation, VIII:CAg was eluted at the void volume together with VIIIR:Ag whereas after coagulation of the plasma VIII:CAg devoid of VIIIR:Ag was eluted after the void volume. Similarly, when the supernatant plasma from blood was chromatographed before the onset of coagulation, VIII:CAg together with VIIIR:Ag was eluted at the void volume whereas during and after coagulation the amount of VIII:CAg associated with VIIIR:Ag gradually decreased. However, no concomitant increase of the concentration of dissociated VIII:CAg was noted under the latter conditions. It seems likely, therefore, that adherance of dissociated VIII:CAg to cellular constituents accounts for the loss of VIII:CAg during coagulation of blood. On the other hand, it can not be excluded that cellular enzymes, extruded during coagulation, affect the antibody-binding capacity of VIII:CAg.

Further studies indicate that, at least in part, dissociation of the factor VIII:VWF complex during coagulation is mediated by thrombin.
Immunological Studies Of Human Factor XI
Authors: Bouma B N , Vlooswijk R A A , Griffin John H
Published: July 2018

Human Factor XI was purified from normal plasma, using ionic exchange chromatography on DEAE-sephadex at pH 8.3, and SP-sephadex at pH 5.3., followed by an affinity chromatography column of insolubilized high M.W. kininogen. Factor XI adhered to this column and was step eluted with 0.5 M NaCl. This rapid method gives highly purified Factor XI with yields of 15-20%. Antibodies were raised in goats against purified Factor XI. A trace of a contaminating antibody was removed by absorption with a Factor XI-depleted γ-globulin fraction. When tested by double immunodiffusion, one precipitin line was observed against normal plasma, which showed a reaction of identity with the line obtained against purified Factor XI. No line was observed against plasma of a patient with Factor XI deficiency. The antiserum specifically inhibited the Factor XI clotting activity of normal plasma.

Factor XI, which is a γ-globulin (pI ∼ 9) that does not migrate during immunoelectrophoresis (IEP), forms reversibly complexes with high MW kininogen, an a-globulin. Complex formation under conditions of IEP at pH 8.4 was shown in purified mixtures by crossed IEP using anti Factor XI and by rocket IEP. Rocket IEP showed no Factor XI antigen in Factor XI deficient plasma. Factor XI antigen in normal plasma was increased by addition of high MW kininogen, suggesting that part of the Factor XI present in normal plasma is not complexed to high MW kininogen. These results show that IEP studies of Factor XI and high MW kininogen are influenced by complex formation.
Influence of operative trauma on F XII and Inhibitor of plasminogen activator
Authors: Hedner U , Bergqvist D
Published: July 2018

A parallel decrease of factor XII and an inhibitor of plasminogen activator (PA inhibitor) earlier described by Hedner (1973, 1980) was observed in a small series of patients postoperatively (Hedner 1978). In order to further investigate this, 52 patients (29 males and 23 females) were studied in connection with vascular surgery. The following parameters were analysed: factor XII (clotting and imm.chem.assays), PA inhibitor, α2-antiplasmin (α2AP), α2-macroglobulin (α2M) and CT-inactivator (CT-INA)(rocket technique) and factor IX (clotting assay). A significant parallel decrease to about 50 % of factor XII measured with both methods reaching its minimum on day 4-5 postop as well as of the PA inhib was seen. This decrease was found in all patient groups except those operated upon because of varicose veins. These latter patients all left the hospital already on the 2nd or 3rd postop day. No concomitant decrease of α2AP, α2M, CT-INA was seen. The observed lowering of f XII and PA inhib on day 4-5 did not depend on changes in haematocrit associated with the operation.

The findings of a parallel decrease of f XII and the PA inhib shown to inhibit f Xlla (Hedner and Martinsson 1978) may indicate that an activation of f XII followed by consumption of this factor as well as of the inhibitor to f Xlla occurs during or after surgery. Whether the f XII dependent fibrinolysis is of importance in the defence against deep venous thrombosis is still debatable. It is, however, striking that low levels of f XIIa occurs postop at a time when most proteins show an increased synthesis rate because of stimulation by the acute phase reaction. Furthermore inhibitors against the activation phase is known to be associated with thrombotic disease (Hedner and Nilsson 1976).
Detection Of Hypercoagulable State By Measuring Mechanical Impedance Of Clotting Whole Blood
Authors: Sugiura K , Handa M , Yoshii Y , Ikeda Y , Toyama K , Ono F , Watanabe K , Ando Y
Published: July 2018

The transition of mechanical impedance of clotting whole blood was determined by Sonoclot(Scienco Inc. U.S.A.) in 22 normal individuals, 14 patients with arterial thrombosis and 20 patients with venous thrombosis. Sonoclot primarily senses loss modulus and records entire coagulation process continuously. Peak g represents the maximum mechanical impedance of clot and slope i represents the rate of gelling process. Sixty-five per cent of cases with venous thrombosis and 36 % of cases with arterial thrombosis showed abnormally high values either in g or in i. In venous thrombosis, there were positive correlations between hematocrit and i, and between fibrinogen and g (r=0.6, p<0.01)In arterial thrombosis, there were positive correlations between platelet count and g (r=0.6, p<0.05) and negative correlations between fibrinogen and g and also between fibrinogen and i (r=-0.6, p<0.05). In normal individuals, no significant correlations were noted between any two of these parameters.

Our results suggest that analysis of clotting process from the biophysical aspect is important to demonstrate hypercoagulable state and that mechanical impedance can be a common indicator of hypercoagulable state.
Inhibition Of Platelet Aggregation, Malonyldialdehyde And Thromboxane Formation By Hydroperoxides Of Arachidonic Acid
Authors: Aharonv D , Smith J B , Silver M J
Published: July 2018

The arachidonate hydroperoxides 12-HPETE and 15-HPETE were biosynthesized from arachidonic acid using partially purified human platelet lipoxygenase or soybean lipoxidase respectively, and isolated by thin layer chromatography. Both compounds inhibited the arachidonic acid- induced aggregation of washed human platelets, suspended in calcium-free Krebs Henseleit solution, in a dose dependent fashion at concentrations between 1 and 50 uM. No inhibition was seen with up to 100 uM of these hydroperoxides when platelet -rich plasma was used. 12-HPETE (in micromolar concentrations) inhibited the formation of both thromboxane B2 (radioimmunoassay) and malonyldialdehyde (spectrophotometrie assay) when washed platelets were incubated with arachidonic acid. The 12-hydroxide, 12-HETE also inhibited platelet aggregation and thromboxane formation, but was less potent than 12-HPETE. We suggest that arachidonate hydroperoxide generated in platelets via the lipoxygenase pathway modulates platelet aggregation induced by arachidonic acid by inhibiting thromboxane formation.
Extracellular Calcium And Platelets
Authors: Taylor P M , Heptinstall S
Published: July 2018

To gain more information on the role of extracellular Ca in platelet behaviour, the movement of 45Ca between plasma and platelets has been studied. Ttoo experimental procedures have been used: platelets were either studied in plasma that contained near-physiological levels of divalent cations or were studied in divalent cation-depleted plasma.

There was a continuous movement of Ca from plasma into platelets when the latter were suspended in plasma that contained near-physiological levels of divalent cations. The iptake was linear with time (2.0 to 2.5 ng ion Ca/109 platelets/60 mins) and was faster at 37°C than at 25°C. The amount of Ca taken up by the platelets increased as the extracellular Ca level was increased and was markedly inhibited by Mg. Sr did not affect the uptake. EGTA displaced only a small amount of the Ca that associated with the plater lets which indicated that Ca was taken up into an intracellular pool rather than sinply bound to the platelet surface. The relevance of this movement of Ca into the cells to platelet behaviour has not been established.

Studies using platelets suspended in divalent cation- depleted plasma shewed that extracellular Ca was in equilibrium with Ca bound at or near the platelet surface. The binding of Ca was time-dependent but saturable (0.30 to 0.50 ng ion Ca/109 platelets/30 mins), and the majority was readily displaced by EGTA. The amount of Ca bound to the cells increased as the extracellular Ca level was increased but was little affected by an excess of either Mg or Sr. Mare Ca bound to platelets when they were incubated at 25°C than at 37°C. This was because platelets lost their ability to bind Ca when they were incubated at 37°C in divalent cation-depleted plasma. This phenomenon was time-dependent and irreversible and was paralleled by a loss in the ability of the platelets to aggregate. These Ca binding sites would seem to be relevant to the aggregation process.
The Role Of Membrane-Bound Tubulin In Platelet Functions
Authors: Ikeda Y , Handa M , Yoshii Y , Imai M , Sugiura K , Toyama K , Watanabe K , Ando Y
Published: July 2018

Evidence has been presented which suggests the existence of tubulin, subunit protein of microtubules, as an integral part of plasma membrane of certain cells. We have investigated whether tubulin is also a constituent of platelet plasma membrane or not, and, if so, what the functional significance is? Platelet membranes isolated by glycerol lysis technique according to the method of Barber and Jamieson retained colchicine-binding activity, 6.2 ± 1.4 n mol colchicine per 100 mg platelet membranes. Colchicine-binding activity of platelet membranes was not decreased after membranes were washed 3 times, indicating that colchicinebinding activity of membranes is not due to contamination of loosely bound cytoplasmic soluble tubulin. On SDS-poly- acrylamide gel electrophoresis, platelet membranes revealed Coomassie blue stained band of molecular weight 55,000, which comigrated with purified cytoplasmic tubulin isolated from human platelets by two successive cycles of temperature -dependent polymerization depolymerization as described previously(Ikeda & Steiner, J. Biol. Chem. 251:6135, 1976). Monospecific antibody against platelet tubulin was prepared in rabbits by injecting soluble tubulin at weekly intervals for 4 weeks. Platelets preincubated with anti-tubulin F(ab’)2 fragment showed reduced platelet aggregation and shape change induced by collagen, but not by ADP or epinephrine. Collagen-induced release of 14C-serotonin was also inhibited by anti-tubulin F(ab’)2 fragment while ADP- or epinephrine-induced serotonin release was not inhibited(collagen 2μg/ml:45.6% of control, ADP 10μM:92.0% of control, epinephrine 4μg/ml: 98.0% of control).

Our results suggest that membrane-associated tubulin may play important roles in collagen-platelet interactions.
Is Phospholipase A2 (PLA2) Involved In PAF-Acether Formation By Platelets
Authors: Chignard M , Vargaftig B B , Le Couedic J P , Benveniste J
Published: July 2018

PAF-acether (platelet-activating factor) has been recently identified as l-O-alkyl-2-acetyl-sn-glyceryl-phosphorylcholine, and later chemically synthetized. Platelets form PAF-acether upon stimulation with the calcium ionophore A 23187 or with more physiological stimuli such as thrombin or collagen. By contrast, arachidonic acid (AA) and adenosine diphosphate (ADP) do not trigger formation of PAF-acether. Since 1) PAF-acether is a phospholipid derivative and 2) aggregating agents which trigger PAF-acether formation are potent platelet PLA2 stimulators, we speculated that PLA2 could be implicated in its formation.

Rabbit washed platelets were incubated at 37°C in the presence of thrombin (2.5 U/ml) or of ionophore A 23187 (2.5 uM) for 7 min and ethanol (80 % final) was added. After centrifugation, the supernatant was evaporated and concentrated. The extract was tested for its aggregating property on rabbit washed platelets preincubated with a cyclo-oxygenase inhibitor (aspirin) and an ADP scavenging system (creatine phosphate and creatine phosphokinase).

In the presence of calcium chelating agents such as EDTA (5 mM) and EGTA (5 mM) most of the synthesis of PAF-acether was suppressed (93 % and 100 % of inhibition respectively). Dibutyryl cyclic AMP (5 mM) also suppressed PAF-acether formation from platelets challenged by thrombin or by the ionophore A 23187 (100 % and 62 % inhibition respectively). Bromophenacyl bromide (0.1 mM) and compound CB 874 (0.1 mM) proved also to be very potent inhibitors of PAF-acether synthesis (100 % inhibition both). All these drugs are well-known platelet PLA2 inhibitors. Upon stimulation platelets also form a deacetylated PAF-acether (lyso- PAF-acether) which could be the direct precursor of PAF-acether. The release of lyso-PAF-acether and the blockade of PAF-acether formation by various molecules having in common a PLA- inhibitory activity lead us to conclude that a PLA2 may be implicated in PAF-acether formation from platelets. Alternative explanations include the possibility that the various inhibitors act on other membrane-related sites.
Therapeutic Trial Of Prostacyclin In Thrombotic Thrombocytopenic Purpura (TTP)
Authors: Budd G Thomas , Bukowski Ronald M , Lucas Fred V , Cook Linda F , Cocchetto David M
Published: July 2018

Deficiency of a plasma factor stimulating vascular prostacyclin (PGI2) generation has been implicated in the pathogenesis of TTP, raising the possibility that PGI2 replacement therapy might be useful in the treatment of TTP. A diagnosis of TTP was made in a 47-year old woman presenting with weakness, fever, and fluctuating neurologic signs. Initial hematologic values were: hemoglobin 7.3 gm/dl, platelets 8000/mm3, reticulocytes 23%. Peripheral smear demonstrated marked microangiopathic changes. Initial therapy was with 8 plasmaphereses, each consisting of a 3 liter exchange. Fresh frozen plasma was the replacement solution in all but the last 2, where 5% plasma protein fraction was substituted due to a suspected plasma hypersensitivity reaction. Additionally, prednisone, aspirin, and dipyridamole were administered during the last 3 plasma exchanges. Transient increase in platelet count occurred but returned to 9000/mm3. Neurologic symptoms remained unchanged. A continuous I.V. infusion of PGI2 was then initiated, and used alone for days 1-5. Aspirin and dipyridamole were added during days 6-8. Prior to use, each PGI2 preparation was assayed for in vitro ability to inhibit ADP-induced platelet aggregation. The average PGI2 infusion rate was 1.9 ng/kg/min for days 1-5 and 3.8 ng/ml/min during the final 3 days. Toxicity included nausea, vomiting, and headache and prevented further dose escalation of PGI2. Platelet count during the 8 days of PGI2 ranged from 5,000-30000/mm3. The patient expired on day 8 due to intracerebral hemorrhage. Postmortem examination demonstrated widespread microthrombi in multiple organ sites. PGI2 infusion in the dosage range employed was a therapeutic failure in this case of TTP refractory to conventional therapy; further trials are in progress.
Prostacyclin In The Treatment Of Raynaud’s Syndrome
Authors: Belch J J F , Newman P , Drury J , Leiberman P , Forbes C D , Prentice C R M
Published: July 2018

There is evidence to suggest that platelet activation occurs in Raynaud’s syndrome in the form of raised β-thromboglobulin and enhanced ADP induced aggregation. We evaluated the effect of prostacyclin (PGI2) in 5 female patients with Raynaud's syndrome. Out-patient visits were made at weekly intervals for 4 weeks. At the first visit buffer solution (Wellcome Laboratories) was infused intravenously for 5 hours, thereafter three five hour infusions of PGI2 at a peak dose of 10 ng/Kg/min were given. Six weeks after the infusions patients were reviewed. Symptomatic improvement including healing of ischaemic ulcers occurred in 4 out of 5 patients. Thermography of the hands confirmed an increase in hand temperature in 3 patients. No increase in temperature was observed in the one patient who did not improve symptomatically, nor in another patient who improved clinically but who had initially inflammation of the hand. Nausea and headache were experienced by all patients but only one patient required a decrease in PGI2 dose to 7.5 ng/Kg/min. We conclude that further evaluation of PGI2 in the treatment of Raynaud’s syndrome is warranted, especially as the duration of improvement far exceeded the duration of treatment.
Platelets, Dietary Fats, And Thrombosis. International Pilot Epidemiological Study In Thrombosis
Authors: Carvalho A C A , Iacono J M , Galli C , Paoletti R , Keys A
Published: July 2018

The influence of dietary fats on plasma, platelet lipid composition and platelet behavior was studied in subjects from 3 countries with different mortality rates from coronary heart disease. Sixty male farmers, age 40-45, from Finland, Italy and the USA were selected. The Finnish diet was the highest in saturated fats and the American in poly-unsat. fats. The Italian diet was the lowest in sat. fats, but 14-fold higher than the other diets in its alcohol content. Plasma cholesterol levels were highest in the Finns (241g%), lowest in the Americans (191g%), and intermediate in the Italians (213g%). Plasma phospholipid (PI) level was high in the Italians and Finns but low in Americans(p< 0.001). Analysis of platelet-phospholipids showed that phosphatidylethanolamine was highest in the Finns, phosphatidylcholine was lowest in the Americans, and phosphatidylserine did not differ among the groups. Sphingomyelin and phosphatidylinositol (PI) were low in platelets of the Finns. However, the content of linoleic acid was highest in the platelet-PI fraction from the Finns. Fatty acid composition of plasma and platelet-PL generally reflected that of the diets. Platelets from the Finns were hyperreactive to epinephrine and thrombin in comparison with the other groups (p<0.001). Platelets from the 3 groups produced similar amounts of thromboxanes from 14C-arachidonic acid, but those of the Finns generated more lypoxygenase derivatives. The latter products may suppress PGI2 production by the vessel wall rendering the platelets more susceptible to aggregation in vivo. Our data show that a diet rich in sat. fats is associated with changes in platelet-lipid composition and platelet behavior. Taken together, these abnormalities cannot be ignored as potential contributing factors to the excessive morbidity and mortality by coronary heart disease found in the Finnish population.
Enhanced Phospholipase A2 Activity In Cholesterol-Enriched Platelets From Rabbits Fed A Cholesterol-Enriched Diet
Authors: McLeod A J , Johnson M , Sucklino K E , Walton P
Published: July 2018

Phospholipase A2(PLA2) could be the rate-limiting enzyme in the metabolism of arachidonic acid (AA) derived from membrane phospholipid to thromboxane A2 (TXA2). Mal- ondialdehyde (MDA) production, which is considered to be an index of TXA2 synthesis, is increased in platelets which have been enriched in cholesterol by incubation with cholesterol-rich phospholipid dispersions in vitro.

Rabbits were fed a diet supplemented with 0.5% w/w cholesterol for 4 weeks after which serum cholesterol was determined and the platelets examined and compared with rabbits fed a control diet. The cholesterol:phospholipid molar ratio (c/p) in the platelets, MDA production (stimulated by AA (imM) and basal) and PLA2 activity were estimated. PLA2 activity was estimated by measuring the % inversion by resuspended washed platelets of 1-acyl-2-(l- 14C)arachidonyl phosphatidylcholine to (1- 14C)arachidonic acid on stimulation with collagen (2μg/ml). Serum cholesterol in the cholesterol-fed group (n=9) was 488 ± 104 mg/ 100ml compared with the control group (n=3) which was 34 ± 4.7 mg/l00ml. Platelets from the cholesterol-fed rabbits showed a 20% increase in C/P (p<0.05); basal and AA stimulated MDA production was increased by 40% and 27% respectively compared with platelets from the control group. PLA2 activity was 1.26% conversion to products in the cholesterol-enriched platelets compared with 0.10% in the control platelets. This increase in activity was significant (p < 0.05).

These results suggest that increased AA metabolism in cholesterol-enriched platelets may in part be due to increased PLA2 activity. This may reflect a physical effect of cholesterol on the platelet membrane predisposing arachidonyl phosphatidylcholine to PLA2 catalysed hydrolysis.