bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipoprotein Platelet Interaction: The Effect On Platelet Cholesterol Content And Platelet Function
Authors: Aviram M , Brook J G
Published: July 2018

Intracellular cholesterol metabolism is regulated by membrane receptors which selectively bind plasma low density lipoproteins (LDL), the major extracellular source of cholesterol. Human platelets, unlike other cells, are unable to synthesize cholesterol, but bind LDL with specificity. We have shown that very low density lipoproteins (VLDL) and LDL, both of which contain apolipoprotein B compete with 125I-LDL for the platelet binding sites, while high density lipoproteins (HDL) is only able to compete to a limit extent and this by virtue of its apolipoprotein E content. Platelet uptake of both LDL and HDL is saturable at physiologic concentrations of these lipoproteins. The lipoproteins are internalized by the platelet but degradation occurs only to a limited extent. Incubation of LDL AND HDL with gel-filtered platelet results in significant changes in the platelet cholesterol content. IDL ( 1 mg protein/ml) increases cholesterol content by 15% whereas the same concentration of HDL causes a 5% reduction. In the presence of thrombin LDL enhances platelet aggregation by 300% whereas HDL decreases aggregation by 50%.

We have thus shown that the lipoprotein platelet interaction alfects both platelet cholesterol content and also platelet aggregation. LDL and HDL have opposing effects and this again highlights their different roles in the atherogenic process.
Cohort Labelling Of Rat Platelets With 7 5Se-Methionine
Authors: Martin J F , Francis Prudence , Penington D G
Published: July 2018

The origin of platelet density heterogeneity is in dispute. We examined whether this heterogeneity is age-related or whether platelets of differing density are initially produced from megakaryocytes. 75Se-methionine labels megakaryocyte protein and hence platelets produced after its intravenous injection. Rats were injected intravenously with 30 μCi of 75Se-methionine. On each of the succeeding 5 days blood was taken from groups of 5 rats following intravenous injection of Prostaglandin E1 (PGE1), and platelets isolated by velocity sedimentation into gradients of polyvinylpurrolidone coated colloidal silica (Percoll).

The isolated platelets, which represented > 93% of the whole blood population and showed < .002% leukocyte contamination, were spun to equilibrium through continuous linear gradients of Percoll. Fractionation of these gradients yielded 15 density dependent platelet subpopulations, whose platelet count and density were measured.

After three washes with cold isotonic saline containing PGE1, each fraction was also counted for platelet associated 75Se activity.

The relationship of radioactivity to platelet count for each fraction for each day was measured by the mean index of concordance which was between 9.997 and 9.992 for days 1 to 5.

On day 2, modal radioactivity and modal platelet counts were found at mean densities of 1.0674 g/ml and 1.0682 g/ml respectively.

It is concluded that platelet density does not vary with platelet age.
Effect Of Selective Inhibition Of Platelet Thromboxane On Platelet-Vessel Wall Interaction In Vivo
Authors: Chen Y C , Wu K K , Hall E R , Venton D L , Le Breton G C
Published: July 2018

It is well recognized that thromboxane A2(TXA2) plays an important role in platelet reactivity. To determine the role of TXA2 in platelet-vessel wall (P-V) interaction, the effect of 1-benzylimidazole (1-BI), a specific inhibitor of thromboxane synthetase, and 13-azaprostanoic acid (APA), a TXA2 antagonist, on platelet thrombus formation was evaluated in vivo in NZW male rabbits using the autologous indium-111 (111In) labeled platelet technique. Rabbits were treated with intravenous 1-BI or APA or vehicles. After injection of autologous 111In-platelets, de-endothelialization of the abdominal aorta was created by a balloon catheter technique. At 3 hrs, blood samples were obtained and the animals were sacrificed. The aortae were removed and the injured and uninjured segments were dissected. Radioactivity counts and dry weight of the tissues and blood were determined. The vascular radioactivity counts were converted to platelet numbers by using a standard linear calibration curve. As small numbers of platelets adhered to normal vessel wall nonspecifically, this number was subtracted to obtain specific platelet accumulation at the injured sites. 1-BI at 10mg/kg reduced the specific platelet accumulation significantly (n=5, 12.3±S.D.I.5×106 pl/gm tissue; p<0.01) when compared with the controls (n=10, 33.0±5.1×106 pl/gm tissue). Platelet accumulation was further reduced by increasing the dosage to 30mg/kg. By contrast, APA injection (10mg/kg) had no significant effect. However, when APA was given by constant infusion at 250μg/kg/min 1 hr prior to injury, the APA-treated animals had an 80% reduction of platelet accumulation relative to controls. These findings indicate that TXA2 plays an important role in P-V interaction and specific inhibition of TXA2 appears to be efficacious in eliminating platelet thrombus formation.
Controlled Studies On The Pathogenesis Of Microvascular Endothelial Damage In Hyperacute Cardiac Allograft Rejection
Authors: Forbes R D C , Guttmann R D
Published: July 2018

Widespread platelet aggregation and microvascular endothelial damage are characteristic features of the early post transplant phase of hyperacute rat cardiac allograft rejection. Vascular staining for rat C3 and rat IgG are consistent immunohistochemical correlates of these alterations. In order to determine the role of platelets and of complement in the pathogenesis of microvascular destruction in hyperacute rejection, a series of ACI rat cardiac allografts and LEWIS (LEW) syngeneic heart grafts transplanted to ACI skin presensitized LEW recipients were studied over the initial 15 minutes posttransplantation. Intravenously administered colloidal carbon was utilized as a tracer for detection of vascular damage by combined light and electron microscopy. All allografts in unmodified combinations showed extensive platelet aggregation and microvascular endothelial damage. Allografts transplanted to recipients in which C3 had been effectively depleted by cobra venom factor showed no microvascular alterations and were comparable to syngeneic controls. There was no depression of recipient platelet counts in the C3 depleted group at the time of transplantation. Allografts transplanted to recipients in which a profound thrombocytopenia had been induced as a result of prior administration of a rabbit antirat platelet globulin preparation showed extensive microvascular destruction at 15 minutes posttransplantation. Recipient platelet depleted syngeneic heart grafts showed no vascular alterations and were comparable to unmodified syngeneic controls. There was no depression of serum hemolytic C3 in platelet depleted recipients at the time of heart graft placement. This study indicates that complement activated by donor specific alloantibodies may directly induce endothelial damage in hyperacute rat cardiac allograft rejection and that platelets are not critical initiators of this process.
Damage To Endothelial Cells In The Ear Artery Of Rabbits On An Atherogenic Diet
Authors: Silver M J , Sedar A W , Ingerman-Wojenski C M , Nissenbaum M , Klerfeld D , Kritchevsky D M
Published: July 2018

Male New Zealand rabbits were fed a diet containing 2% cholesterol, 6% peanut oil and 92% Purina Chow for periods between 6 and 9 weeks. Examination of the inner surface of the central ear artery by scanning electron microscopy (after perfusion in situ with Tyrode’s solution followed by 1% glutaraldehyde) revealed damage to the endothelium which appeared to be considerably worse at 9 weeks than at 6 weeks. At six weeks the damage included irregularly shaped cells, some breaks at intercellular junctions and occasional holes in the cells. At 9 weeks more severe damage was seen. This included many misshapen cells, cells with holes, and many cells lifting off from the vessel wall and beginning to expose sub-endothelial tissue. In 3 rabbits (respectively on the diet for 6,7 or 9 weeks) arrays of dark “spots” were seen in many cells. Such “spots” were never seen in endothelial cells of normal rabbits. After 1 ml of 0.83 mM sodium arachidonate (AA), instead of Tyrode’s solution, had been perfused through the arteries, further damage was seen. This damage included the lifting off of many cells and formation of many holes. Effluent from the arteries perfused with AA contained 6-keto-PGF1α and TxB2 (measured by specific radioimmunoassays) in amounts similar to those produced by arteries of rabbits fed a normal diet. These metabolites were not detected in effluents of arteries perfused with Tyrode’s solution.

This report shows that an atherogenic diet may cause early morphological changes in a peripheral artery of the rabbit and suggests that widespread damage to arterial endothelium may result from atherogenic diets. Apparently, this damage does not impair the ability of the vessel wall to convert AA into prostacyclin. The rabbit ear artery system appears to be a simpler way to study the early changes in arterial endothelium during experimental atherosclerosis than studies on the aorta.
Role Of Surface Negative Charge In Platelet Aggregation
Authors: Tanoue K , Jung S M , Isohisa I , Sakakibara C , Ariga S , Yamazaki H
Published: July 2018

The relationship between aggregability and electrophoretic mobility (EPM) was studied to clarify the role of surface negative charge in platelet function. 1. Human platelets were washed 3 times with Phillip’s buffer (pH 7.4), resuspended to 107 platelets/ml in modified Zeiller and Hannig’s buffer with 1 mM EDTA and incubated with various concentrations of ADP, adrenaline, thrombin and ristocetin at 37°C for 3 min. Without further washing, mean EPM of about 700 platelets was determined by the automatic Laser Zee System 3000 with good reproducibility with less than 1 % variation coefficient for 5 measurements. In all experiments, aggregating agents were dissolved in physiologic saline to obtain the same solution conductance both in test and control platelet suspensions to which saline alone was added. Neither 1-100 uM ADP nor 5-500 μM adrenaline had any effect on platelet EPM. Thrombin decreased EPM with dose response relation with -1.692±0.014 μm/sec/V/cm without thrombin, -1.580±0.084 with o725 , -1.578±0.001 with 1.0, -1.454±0.018 with 2.0 and -1.289±0.004 with 5.0 U/ml of thrombin. Ristocetin had decreasing effect on EPM at 0.2 mg/ml or less. After addition of above aggregating agents, the washed platelets did not aggregate under phase contrast microscopy. 2. EPM of washed platelets were compared with aggregability in the PRP collected from 33 clinical cases with various degrees of aggregation. There was no correlation between EPM and secondary and maximum aggregation induced by any of these four agents. However, intensity of primary aggregation by adrenaline correlated with EPM (r= 0.485, P<0.01). The same tendency was observed in primary aggregation by ADP. 3. Treatment of washed rabbit platelets with increasing doses of neuraminidase resulted in increasing aggregation by ADP and collagen associated with decrease in EPM. These findings suggested that platelet surface negative charge may be regulatory role in initiation of platelet activation.
High- And Low-Affinity Heparin Binding Proteins From Bovine Platelets
Authors: Ciaglowski Raymond E , Walz Daniel A
Published: July 2018

The class of platelet-specific proteins which displays anti heparin and/or growth stimulating activities includes: high affinity platelet factor 4 (HA-PF-4), low affinity platelet factor 4 (LA-PF-4) and its closely related variant β-thromboglobulin (β-TG), and platelet derived growth factors) (PDGF). In an effort to determine to what extent these proteins and their activities might be broad-based, a comparative study using bovine platelets has been undertaken. Fresh, washed bovine platelets were either thrombin stimulated or freeze-fractured and the ensuing supernatants chromatographed on either heparin-Sepharose (HS) or dextran- sulfate-Sepharose (DSS). Bovine protein which eluted with either 1.0M NaCl (HS) or 1.5M NaCl (DSS) was subsequently gel filtered to homogeneity. Using S2222 chromogenic assays, the bovine protein (9000 dal tons) and human HA-PF-4 (7800 dal tons) each had similar heparin neutral ization equivalence, about 40 U/mg. Using 3T3 cells, growth promoting activity was not found to be associated with the purified bovine HA- PF-4. Although bovine HA-PF-4 has 15 additional residues from the amino terminal end of human HA-PF-4, the acidic, neutral and basic regions have remained similar. HSand DSS fractions from thrombin released platelets eluted with 0.5M NaCl and gel filtered yielded a homogeneous protein of molecular weight 12,000 dal tons. This product has a heparin neutralization activity of approximately 2 U/mg; cellulose acetate electrophoresis of the bovine protein migrated into the gammaglobulin region, and we have consequently referred to it as bovine LA-PF-4. The bovine LA-PF-4 protein had 3T3 growth stimulating activity at least comparable to that of a DSS intermediate salt eluted fraction. The amino terminal 12 residues of bovine LA-PF-4 are not similar to human LA-PF-4 or β-TG. We conclude that these bovine anti heparin proteins are functionally homologous to their human counterparts. We are currently completing the sequence of bovine HA-PF-4 in order to establish the structure-function relationship of the heparin binding domains of these proteins.
On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation
Authors: Yamamoto M , Watanabe K , Ando Y , Iri H , Fujiyama N , Furihata K , Yoshii Y , Sugiura K , Ikeda Y , Toyama K
Published: July 2018

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.

Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.
The Cyclic-Amp-Lowering Effect Of Pge2 And Of The PGH2 Analog, U46619
Authors: Martin B , Bonne C
Published: July 2018

We have recently reported that PGE2 counteracted the anti-aggregating action of PGE1 and of other PG by inhibiting the adenylate cyclase system. But this effect is not due to the interference with anti-aggregating PG receptors. In this study, the mechanism of action of the c AMPlowering effect of PGE2 has been further investigated.

Suspensions of aspirin-washed human platelets were incubated for 1 min at 37°C in the presence of papaverine (0.5mM) with PGE1 , PGE2, U46619, a chemical analog of PGH2, 13-azaprostanoic acid, a specific antagonist of TxA2, either alone or in various associations. The c AMP concentration was determined by protein binding assays in platelet extracts. PGE2 (l50nM) and U46619 (1μM) inhibited the rise in c AMP induced by PGE1 (30nM) On the other hand, when 13-azaprostanoic acid (507#x03BC;M) was added to the incubate, the inhibitory effects of these compounds were suppressed.

These results support the conclusion that TxA2 and U46619 act on a unique receptor which triggers the c AMP-lowering effect and suggest that PGE2 antagonizes the anti-aggregating PG through interaction with this receptor.
Potentiating Effect Of 5, 8, 11-Eicosatrienoic Acid (20:3ω9) On Platelet Aggregation
Authors: Lagarde M , Burtin M , Sprecher H , Dechavanne M , Renaud S
Published: July 2018

The in vitro effect of 20:3ω9 (5×10-6M) was tested on human platelet suspensions, with aggregation induced by thrombin, ionophore A23187, ADP, collagen and arachidonic acid. When 20:3ω9 was added simultaneously with the aggregating agent, the response was increased by 100 to 300 % with all agents. It was solely to thrombin and ionophore that the aggregation was increased (by 200 to 300 %) when 20:3ω9 was preesterified in the platelet phospholipids by a 2 hour incubation period and subsequent platelet washing. Aspirin at a concentration inhibiting cyclooxygenase activity, did not block the potentiating effect of 20:3ω9. However, that effect was inhibited by a higher aspirin concentration, also inhibiting the peroxydase from the lipoxygenase pathway. Monohydroperoxy (HOO) - and monohydroxy (HO) - 20:3ω9 have been prepared from human platelets and purified by TLC. When added to platelets in vitro, solely the HO-20:3ω9 (0.2×10-6M) increased by several folds the response to thrombin and ionophore. Finally, both thrombin and ionophore induced the formation of HO-20:3ω9 (detected by a radiochemical technique) from platelets enriched with 20:3ω9. Thus, an increase level of 20:3ω9 in the platelet phospholipids seems to markedly enhance the susceptibility of platelets to aggregation by thrombin (and ionophore), probably through the lipoxygenase end product. Consequently the increase in the susceptibility of platelets to thrombin induced aggregation reported in essential fatty acid deficiency as well as with high saturated fat intake, both in man and in animals, might be due to the higher level of 20:3ω9 in the platelet phospholipids, which has been observed under those conditions.
Influence Of Fibrin And Fibrinogen On The Activation Of Plasminogen By Urokinase
Authors: Takada Y , Takada A
Published: July 2018

Human plasma was clotted with thrombin or Ca++ in the presence of urokinase (UK), and the activity of plasmin was measured by hydrolysis of S-2251. As a control, the hydrolysis of S-2251 was measured without the addition of thrombin (in the absence of clot). The hydrolysis of S-2251 was higher in the clot than in the plasma. When euglobulin solution was clotted by thrombin in the presence of UK, the hydrolysis of S-2251 was higher in the presence than in the absence of clot. Thus the increased hydrolysis of S-2251 in the clot after the addition of UK was not due to the interaction of plasmin with inhibitors (∝2AP or∝2M) in the presence of clot, but plasminogen (plg) seems to be better activated by UK in the presence of fibrin. When purified Glu-plg was used, Glu-plg was better activated by UK in the presence of fibrin than fibrinogen. The addition of AP to the mixture of Glu-plg and UK resulted in complete inhibition of plasmin activity but the presence of fibrin partially prevented its inactivation by ∝2AP. When Glu-plg was incubated with plasmin,resuiting Lys-plg was far better activated by UK than Glu-plg, but the presence of fibrin slightly enhanced its activation rate. When fibrinogen or fibrin was degraded by plasmin, early FDP enhanced the activation more than early FgDP. Early FgDP still better activates Glu-plg than fibrinogen. Extensive degradation products (D and E) resulted in less activation of Glu-plg than in the absence of these products. The addition of these degradation products to already activated plasmin had no effect on the enzymatic activity of plasmin.

In conclusion, Glu-plg was better activated by UK when clot was formed. Binding of Glu-plg with fibrin through lysine binding sites protected it from inactivation by AP. Early degradation also resulted in better activation of Glu-plg, but extensive degradation resulted in less activation which may be an important feedback mechanism of fibrinolysis.
Ultrasonic Vibration For Boosting Fibrinolytic Effect Of Urokinase
Authors: Tachibana Shunro
Published: July 2018

Boosting effect of ultrasonic vibration on fibrinolytic activity of urokinase has been investigated. Fibrinolysis was studied grouply in (1) experimental fibrin clot with urokinase, (2) clot with urokinase preceded exposure of clot to ultrasonic waves, (3) clot exposed to ultrasonic waves and (4) clot with saline solution. Scanning electro microscopic examinations were made on the clots in each four series. Fibrinolysis was not evident in the clots (3) and (4), though fine cracks were seen in the clots (3). The four hour rate of fibrinolysis was 50% in the clots (1) where as 100% in clots (2).

Procedures (1)(2)(3) and (4) were also emplyed in the Chandler’s Loop Technique clot of four hour rotation. The rate of fibrinolysis was 50% in the clot (1) when 60 IU of the urokinase was used, where as 86% in the clot (2) , which scanning electro microscopically gave a feature of decreased fiber structure. Fibrinolysis was accere- rated by ultrasonic vibration in the clot with certain amount of urokinase added, breaking up of the clot being reached in shorter time than the series without ultrasonic wave application.

Ultrasonic vibration mechanically might cause microstream in the clot architecture, which could help urokinase contact directly with the fiber net. Scanning electro microscopic examinations revealed morphological representation of enhanced fibrinolysis by ultrasonic vibration. It was suggested that physical ultrasonic vibration could be applied to boost a chemical fibrinolytic effect of urokinase.
On The Mechanism Of Plasminogen Activator Release By Ddavp: Animal Studies
Authors: Boulton F E , Prowse C V , Cash J D
Published: July 2018

In pentobarbitone anaesthetised dogs, IV infusion of 1-desamino-8-D-arginine vasopressin (DDAVP) over 15 minutes produced a dramatic increase in plasma plasminogen activator (PA) levels, as measured by the euglobulin lysis time. However, 70 ug/Kg of DDAVP were required, compared with circa O.4 ug/Kg for a similar response in unanaesthetised human subjects. Factor VIII levels increased only slightly in dogs even at these relatively high doses.

Infusion of 7.0 ug/Kg DDAVP into the femoral artery of dogs had no discernible effects on the PA level of ipsilateral femoral venous blood - similar to findings previously reported for humans. In contrast, infusion of this dose into the carotid artery of dogs produced a marked rise of PA in jugular venous and systemic blood.

Fifty mls of jugular venous effluent were collected immediately after DDAVP infusion to carotid artery, the plasma separated, stored frozen, and subsequently infused intravenously to another dog. PA levels rose moderately.

It is suggested that DDAVP acts on some cephalic receptor - possibly in the hypothalmus - from which a humoral factor is released which in turn releases PA from a peripheral storage site. The high doses of DDAVP required in these studies, and the relative lack of fVIII response, suggests that barbitone anaesthesia may interfere with this mechanism.
Therapeutic Implications Of Heparin Metabolism In Newborns
Authors: McDonald M M , Hathaway W E , Reeve E B
Published: July 2018

While thrombotic complications are relatively frequent in the sick newborn, therapeutic heparinization is often difficult to achieve. The clearance of one bolus dose of heparin has been found to be more rapid in the newborn (T% of 35.5 min. in babies 33-36 weeks gestation vs. 63.3 min. in the normal adult). Decreased levels of antithrombin III (AT-III) in the newborn (20-50% of normal adult) may explain the relatively short T1/2; however, an adult with congenital AT-III deficiency (50% level) was found to have a prolonged plasma heparin T1/2 of 106 min. The function of fetal AT-III was studied by isolating the protein from cord blood of nine term and preterm babies by heparin affinity chromatography. Evaluation by SDS-PAGE, immunoelectrophoresis, heparin cofactor specific activity, progressive neutralization of thrombin and Xa at 37°C, pH related antithrombin kinetics and thrombin neutralization relative to heparin concentration revealed a fetal protein with structure and function indistinguishable from that isolated from normal adults.

Clinical use of heparin included the evaluation of the Laidlaw (LL) micro whole blood clotting time. The mean LL time of 79 newborns was 96.2 sec. (SD=23). The LL time was not related to gestational age (24-42 weeks) nor to size (600-3980 g). Nine babies were heparinized for three days to six weeks for major vessel thromboses. Heparin infusion rates were compared with heparin levels achieved, LL clotting time prolongations and AT-III levels. The newborns required heparin infusion rates of 25 to 40 U/kg/hour to achieve plasma heparin levels of 0.3-0.5 U/ml. The prolongation of the LL correlated with the heparin level achieved. In these nine babies, AT-III levels did not decrease during the course of heparin therapy.

These studies suggest that the newborn has an increased heparin requirement for anticoagulation not completely explained by a low level of a normal antithrombin III.
A Sensitive Heparin-Protamine Competitive Binding Assay
Authors: Dawes J , Pepper D S
Published: July 2018

Using protamine-agarose beads, with 125I- heparin as a tracer, we have developed a sensitive competitive binding assay with very wide applications. We are able to measure the concentrations of heparin and heparan sulphate in protease-treated biological fluids down to 40 ng. ml-1. Chondroitin and dermatan sulphates do not interfere with the assay, which can also be used to:

(1) measure other acidic polysaccharides e.g. dextran sulphate, semisynthetic heparin analogues, and carrageenans.

(2) assay proteins which bind to heparin, such as platelet factor 4, antithrombin III and protamine.

(3) screen polymers and solid phases as potential binding materials for these molecules.

(4) compare a series of polymers e.g. assess the degree of sulphation of heparins, or the affinity of basic polymers for heparin.

Solid phase polyelectrolytes such as Dowex are potentially more robust substitutes for protamine- agarose, being resistant to proteases.
Heparin Associated Thrombocytopenia Is Not A Typical Drug Induced Thrombocytopenia
Authors: Kelton J G , Powers P J , Turpie A G G , Carter C J
Published: July 2018

Recent studies have suggested that heparin associated thrombocytopenia (H.A.T.) is mediated by immune mechanisms. We studied 7 patients who developed thrombocytopenia while receiving heparin therapy. All of these patients had elevated platelet associated IgG (PAIgG) at the time of the thrombocytopenic episode which ranged from 6 to 47 fg/IgG per platelet (normal less than 5). In all patients the PAIgG returned to normal when the platelet count normalized. Ninety non-thrombocytopenic patients receiving heparin had normal PAIgG on multiple determinations. In four of the H.A.T. patients, recovery serum plus test platelets were incubated with varying concentrations of heparin (0.01-10 I.U. per ml) and the level of PAIgG measured on washed platelets. The PAIgG on the test platelets was within the normal range and less than 10 fg IgG per platelet for all heparin dilutions. In contrast, lo of 13 sera from patients with other types of drug induces thrombocytopenia had significant elevation of PAIgG on test platelets when the recovery sera was incubated with the causative drug and test platelets. Two of the four patients with H.A.T. who did not react in vitro to heparin were subsequently re-administered heparin in vivo and they did not develop thrombocytopenia or elevation of PAIgG. The lack of response to either in vitro or in vivo rechallenge indicates that although H.A.T. is associated with increased binding of IgG to platelets at presentation, the mechanism differs from that observed with the more usual drug induced thrombocytopenia.
Development Of Blood Coagulation-A Fetal Lamb Model
Authors: Kisker C T , Robillard J E , Clarke W R
Published: July 2018

To study the normal development of blood coagulation factor activities in a growing fetus while avoiding the effects of labor and delivery, a chronic fetal lamb model was developed in which serial blood samples were obtained from 10 chronically catheterized fetuses during the third trimester of pregnancy and 24 hours following spontaneous delivery of live-born lambs. Under operating room conditions with sterile technique, a polyethylene catheter to which heparin had been bound to both internal and external surfaces, was inserted into the femoral artery of the fetus. The catheter was brought out through a skin pouch to the side of the ewe, and enclosed in a zip lock bag. Blood samples were withdrawn from the catheter three times each week for measurement of coagulation factor activities. Levels of coagulation factor activities at birth in noncatheterized animals were not different from those found in catheterized animals except for factor IX activity which was 12% higher in the catheterized animals (0.02<p<0.05). Fibrin/fibrinogen degradation products and fibrin monomer levels were not increased during gestation and the shape of the changes in activity of individual clotting factors over time in the different fetuses was remarkably constant for each of the factors measured. Fibrinogen, prothrombin, and factor VII showed a decrease in activity early in the third trimester of pregnancy, whereas factors V, VIII, IX, X, XI, XII, and XIII showed a gradual increase in activity throughout the last trimester of pregnancy. Both factors VIII and IX showed a significant increase in activity (23% factor VIII and 12% factor IX) associated with the process of delivery. Changes in blood coagulation in the fetal lamb were similar to those thought to occur in humans. Similarities included fibrinogen, factors V and VIII which reached or exceeded adult levels at birth and the vitamin-K dependent factors II, VII, IX and X which remained below adult levels at term.
Evaluation Of Thigh Length Sequential Leg Compression In The Prevention Of Deep Venous Thrombosis
Authors: Hartman J T , Yost R P , Robertson W W , Janssen H F
Published: July 2018

The present study is designed to evaluate the effectiveness of a thigh length sequential compression device (SCD) in the prevention of deep venous thrombosis (DVT) in patients undergoing hip operations (either fracture or elective). The night before the operation phleborheography, doppler ultrasound and, when possible, 125I fibrinogen scanning were used to evaluate venous flow in the patients’ legs. The patients identified as being free of DVT complications were then fitted with the compression sleeves. Following surgery the venous evaluation tests were repeated on alternate days to determine if DVT complications had developed. Evidence of DVT formation was identified in 10 of the 52 control patients (who received leg elevation only) and in 1 of the 52 treated patients (who received the SCD plus leg elevation). The chi square statistic demonstrates a significant difference between the two groups (P<.05).

Data evaluation indicates that surgical approach and patient obesity did not contribute to the risk of DVT formation. However, patients who are either over the age of 66, females, or have suffered a hip fracture appear to be at greater risk than other groups. The amount of data in this study makes it difficult to evaluate with statistical significance the additional risk produced by other factors such as heart condition, previous history of clotting problems, estrogen therapy, etc.
Thrombectomy For Ilio-Femoral Thrombosis In Fertile Women
Authors: Swedenborg J , Delin A , Hellgren M , Jacobsson H , Nilsson E
Published: July 2018

Thirteen women, age 29-48 years, with iliofemoral venous thrombosis were subjected to thrombectomy via the femoral vein, and construction of an arteriovenous fistula. Anticoagulant therapy was given from the operation and 6 months on. The AV fistula was closed after 3 months. After cessation of anticoagulant therapy the patency of the iliac vein was evaluated by plethysmography and, if this was inconclusive, by phlebography. A work-up for coagulation abnormalities was also made.

All thromboses were left-sided. Several risk factors were present: pregnancy in 9 of 13 (2 early) contraceptive pills in 2 of 13, and iliac vein stenosis in 9 of 10. In 8 of 12 decreased levels of plasminogen activators were noted. Of the 9 pregnancies, three were complicated and required interruption, one was terminated by normal delivery one week before thrombectomy, two were interrupted at the time of thrombectomy, and three continued to normal deliveries several weeks after thrombectomy.

No pulmonary embolism was observed. At follow up, the iliac vein was patent in eleven patients. It is concluded that the pathogenesis of iliofemoral venous thrombosis in fertile women is multifactorial, involving hormonal and mechanical components. This study draws attention to decreased fibrinolytic activity in many of the patients. Thrombectomy resulted in iliac vein patency in eleven of thirteen patients. The procedure is well tolerated by mother and child during pregnancy.
Monitoring Low-Dose Heparin Administration In Hip Surgery
Authors: Poller L , Tabemer D A
Published: July 2018

Fifty-five patients requiring selective hip replacement or emergency surgery for hip fractures were randomly given fixed low-dose subcutaneous calcium heparin 5,000 units 8-hourly (30 patients) or monitored subcutaneous calcium heparin (25 patients). The aim was to prolong the activated partial thromboplastin time (APTT), using the NRLARC method, to 5 seconds above the upper limit of normal.

Adjusting the dose of heparin was moderately successful in achieving the target value for the APTT (46% of observations) compared to the fixed dose group (27%) p<0.005. In nine patients prophylaxis failed to prevent DVT detected by 125I-fibrinogen scan; three were in the adjusted dose, six in the fixed dose heparin group. In all nine patients the APTT showed less than the desired prolongation the day before the scan became positive although in six patients with positive scans, measurable heparin levels were detected by anti-factor Xa assay. The APTT appears, therefore, to give a better guide during hip surgery to the antithrombotic effect of heparin than the anti-factor Xa assay in low-dose heparin prophylaxis. Maintaining the APTT at or above 50 seconds with the NRLARC method protected these high risk patients from post-operative DVT.
Histochemical Studies On The In Vitro 5-HT Uptake In Endothelial Cells
Authors: Kjellström T , Ahlman H , Dahlström F , Hansson G , Stenberg B , Risberg B
Published: July 2018

Previous studies have shown that 5-HT is rapidly taken up by the endothelial cells and some investigations also suggested that serotonin is metabolized within these cells. In earlier studies on rat-lungs using a fluorescence histo- chemical method according to Hillarp - Falk we demonstrated that 5-HT was accumulated within the mast cells. Using this technique we could not demonstrate any specific uptake in the pulmonary endothelial cells. It was the purpose of the present investigation to further study the 5-HT uptake by isolated pulmonary endothelial cells.

Methods Cells from the vascular intima of the pulmonary artery in adult rats were grown in a growth medium containing FCS. The endothelial nature of these cells was demonstrated using transmission electron microscopy and factor VIII analysis. Confluent endothelial cells were incubated with 5-HT and the cellular uptake was studied with fluorescence microscopy according to the Hillarp - Falk procedure.

Results The endothelial cells were identified by the presence of Weibel-Palade bodies using transmission electron microscopy and the immunofluorescent demonstration of cellular factor VIII antigen. Cells not exposed to serotonin had no specific 5-HT fluorescense. After incubation with 5-HT at different concentrations there was a progressive uptake of the amine within the cells.

Conclusions This study confirms previous reports on the specific serotonin uptake in endothelial cells. The Hillarp-Falk procedure seems suitable for further studies of serotonin uptake in cultured endothelial cells.
The Effect Of Ticlopidine On Cultured Human Endothelial Cells: Possible Influence On Extracellular Matrix Formation
Authors: Piovella F , Piovella L , Almasio P , Ricetti M M , Ascari E
Published: July 2018

Endothelial cells derived from human umbilical cord vein were seeded and grown to confluence in culture medium TC 199 with 20% foetal calf serum without other growth factors supplement. Cells were then detached utilizing a trypsin-EDTA solution, harvested in fresh culture media containing different concentrations of Ticlopidine, poured on glass coverslips placed on 35 mm Petri dishes and cultured for six days without further medium replacement. Control dishes were prepared using fresh medium without Ticlopidine.

Treated cells revealed a decreased tendency to adhere to the substratum as well as morphological changes in comparison with controls when examined by phase contrast microscopy. This effect was more evident with higher concentration (0.33 mg/ml) of Ticlopidine, while lower concentrations (0.05 mg/ml) caused morphological changes only. Indirect immunofluorescence for fibronectin utilizing monospecific antibodies, revealed a different fluorescent pattern if compared with controls. In particular a less pronounced intracellular granular fluorescence and a delay in the appearance of the fibrillar extracellular matrix.
Characterisation Of Vaccinium Myrtillus Antho-Cyanosides Effect On Cultured Human Endothelial Cells
Authors: Piovella F , Almasio P , Ricetti M M , Ascari E
Published: July 2018

Vaccinium myrtillus anthocyanosides (Vma) have been demonstrated as significantly effective substances in promoting wound repair and the formation of new capillaries in experimental animals. Their effect have been also studied in clinical trials,
demonstrating a significant anti-haemorrhagic action in patients with defective platelet-endothelium axis (primary haemostasis). We report the results of an extensive study performed on human endothelial cells (h.e.c.) derived from umbilical cord vein and cultured in vitro in the presence of various Vma concentration. As controls non-treated h.e.c. as we 11 as dexamethasone-treated h.e.c . were used. H.e.c. growth rate as well as their morphology, factor VIII related antigen (FVIII R:Ag) content and release and fibronectin extracellular matrix, were the parameters studied in our experiments.
Vma cause an increased h.e.c. growth in comparison with controls. This action is similar to the effect described for dexamethasone, but it is not mediated by variations of the fibronectin extracellular matrix. Vma-treated cells do not show any variations of FVIII R:Ag immunofluorescent pattern nor significant morphological changes. The stimulatory action on growth rate is extended to the other cell types of the vessel wall as demonstrated by the cultures of suspensions of h.e.c. highly contaminated by fibroblasts and smooth muscle cells.
Arterial Thrombosis In Heparin-Induced Thrombo Cytopenia Is Not Accompanied By A Decreased PGI2 Stimulating Activity In Plasma
Authors: Kulzer R , Schäfer G , Budde U , Popov S
Published: July 2018

A patient developed severe thrombocytopenia and acute arterial occlusion 6 days after onset of heparin therapy. Heparin was ad ministered because of venous thrombosis in the lower limbs. Aggregometry showed the typical signes of heparin-induced thrombocytopenia with all evaluated heparins of different origin. PGI2 stimulating activity of plasma was in the range of normal plasma samples. No aggregating activity of the plasma could be shown without heparin(Method of Lian et al.). Heparin activity was determined before and after aggregation with a chromogenic substrate.

After cessation of heparin therapy the platelets raised into the lower normal range within a few days.
Platelet Factor 4, Heparin Neutralizing Activity And Fibrinogen
Authors: Etherington M D , O’Brien J R
Published: July 2018

To investigate the relationship between the heparin neutralizing activity in platelets (intra-plat HNA = IP HNA) and in plasma (PPP HNA) as measured by the heparin thrombin clotting time (HTCT) and the plasma PF4 (PPP PF4) and the intra-platelet PF4 (IP PF4), we selected 15 people with contrasting amounts of HNA in their plasma; these included young and old controls, patients after operations and myocardial infarcts and pregnant women. Platelet rich plasma (PRP) and PPP were prepared from citrated blood processed entirely at 4°C. 2ml sub-samples of PRP and PPP were then frozen and thawed × 3, centrifuged and the supernatants collected. IP PF4 and PPP PF4 were measured by radioimmunoassay (ABBOTT KIT). To measure IP HNA and PPP HNA a modified Harada and Zucker clotting assay was performed. On each study day PPP was prepared from a control and used as substrate. All test plasmas were added to this PPP and the amount of heparin required to give a clotting time of 30 seconds was measured. IP HNA was calculated by subtraction of PPP units heparin from PRP units. The plasma fibrinogen was estimated by Clauss’ method.

The IP PF4 was significantly correlated with IP HNA (r=0.95, n=15, p<0.001). The plasma PF4 in all volunteers was less than 10ng/ml-1 and bore no relation to the PPP HNA. However the plasma fibrinogen was significantly correlated to PPP HNA (r=0.91, n=15, p<0.001).

Thus the IP HNA accurately reflects IP PF4 (with presumably a parallel small contribution from β-thromboglobulin). The PPP HNA, previously thought perhaps to reflect liberated PF4 or another intra-platelet anti-heparin substance, is now shown to be independent of PF4 but at least in part reflects the plasma fibrinogen (which neutralizes heparin). This quick and simple clotting assay is well suited to a routine laboratory and might be used to measure total IP HNA, and monitor the a granule content of platelets in disease.
Comparison Of The Time Course For Adp-Induced Shape Change, Aggregation And Onset Of Refractoriness
Authors: Milton J G , Glushak C , Wong T , Frojmovic M M
Published: July 2018

Platelet morphology has been classified in our laboratory by phase-contrast microscopy into discocyte (D), disco- echinocyte (DE) and sphero-echinocyte (SE). Following the addition of 1-20 μM ADP to citrated platelet-rich plasma (PRP), platelet morphology changes as D→DE→SE with the maximum in DE occurring at 6-10 sec and that for SE at 1-3 min, and then slowly returns to D over the course of ∼30 min. Here we compare the time courses for early platelet aggregation (PA) and the onset of refractoriness, to the time course for platelet shape change, the latter measured in initially mixed unstirred PRP diluted with plasma to N≤50,000μl-1 to minimize aggregation. PA was measured microscopically as the % decrease in single platelets and refractoriness was measured by aggregometry from ADP dose-response curves determined as a function of time after initial stimulation. The time course for PA was similar to that for the appearance of DE. The DE at the time of maximal PA had pseudopods, but a main body geometry which differed only slightly from that of a D. The time course for onset of refractoriness at 2 μM ADP resembled that for the appearance of SE, indicating that the lifetime for single platelets able to participate in PA is <60 sec. In contrast to ADP, PA induced by 10 μM adrenalin was maximal at ∼ 30 sec and during this time there was little shape change (<20% decrease in D). Thus although PA can occur without shape change, the rate is 3-4 fold faster if shape change occurs. The similarity of the time course and ADP concentration dependence for PA and DE formation suggest an intimate relationship between these processes. Moreover, the above results emphasize the transient nature of single DE participation in aggregation, and in particular point out that the relatively slowly formed ‘sphered’ platelets (SE) are not sticky as single platelets and do not participate in early aggregation.
Glutathione Peroxidase And Malondialdehyde Production In The Arachidonic Acid Metabolism Of Human Platelets
Authors: Guidi G C , Schiavon R , Avventi G L , Perona G
Published: July 2018

A series of functional parameters, including the aggregability triggered by various agents, the in vitro malondialdehyde production and the glutathio ne peroxidase activity, has been investigated in platelets from normal blood donors.

Glutathione peroxidase activity assays showed a significant inverse correlation with malondialdehyde induced by arachidonic acid but not with aggregation data and malondialdehyde induced by thrombin. Moreover, arachidonic acid generates in human platelets lysates large amounts of hydrogen acceptor substrate(s) for the glutathione peroxidase with peculiar kinetic features. These are related to ma londialdehyde production and to partial inhibition by acetyl-salicilic acid and are likely connected with prostaglandin metabolism.

Our data suggest that physiological variations in glutathione peroxidase activity are important in human platelet arachidonic acid metabolism, because they modulate the biosynthesis of key end-products, as thromboxane A2, whose malondialdehyde is an inde.
Clinical Evaluation Of Ticlopidine In The Inhibition Of Platelet Function - A Multiclinic Double Blind Study In Comparison With Aspirin
Authors: Abe T , Kuramoto A , Yasunaga K , Sakuragawa N , Maekawa T , Yamazaki H , Toyama K , Ogawa N
Published: July 2018

Clinical effect of ticlopidine, a new inhibitor of platelet aggregation, at a daily dose of 300 mg was compared with that of aspirin at its daily dose of 500 mg by means of double blind assay. The suppression of platelet aggregations induced by ADP, collagen and epinephrine was compared among platelet samples obtained from 38 cases with thrombotic tendency or at enhanced platelet functions.

The inhibitory efficacy of ticlopidine on platelet aggregations induced by collagen or epinephrine was found as potent as that of aspirin in their overall judgement, but the ticlopidine inhibition on the ADP aggregation was apparently more potent than aspirin. The maximum aggregation rate was significantly decreased and the frequency of the second aggregation pattern was more remarkably minimized in the ticlopidine-treated group than the aspirin-group. The maximum aggregation rates induced by collagen or epinephrine were significantly decreased in both of ticlopidine- and aspirin-groups to the same extent.

Blood filtration velocity through Nuclepore filter of 5μm pore size was significantly enhanced in the ticlopidine- group but no change found in the aspirin-group. Other coagulation parameters and laboratory data were not changed in both groups, except bleeding and prothrombin times which were slightly prolonged in the aspirin-group.

From the results mentioned above, it was expected that ticlopidine might exert superior clinical effect to aspirin on thrombotic diseases and microcirculatory disorders