bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect Of Activated And Non-Activated Protein C On Mouse Tumor Metastases
Authors: Gasic T B , Catalfamo J L , Gasic G J
Published: July 2018

Platelet aggregation (PA) and fibrin deposition appears to be involved in the establishment of blood-borne tumor cells as metastases. Since it has been reported that activated Protein C have fibrinolytic activity or can inhibit coagulation, we investigated whether activated Protein C may inhibit tumor spread. Preliminary studies in the mouse system indicated that (a) activated Protein C prolonged the kaolin cephalin clotting time of plasma treated in vitro (>10min.)or of plasma obtained from in vivo treated mice (55 vs 30 sec.), (b) activated Protein C can inhibit PA by plasma membrane vesicles shed by metastasizing tumor cells; this effect was much greater with rat gel filtered platelets than with heparinized mouse PRP. The inhibition effect was also present in vivo, since 56 ug of activated Protein C, injected ip 15 min. before iv injection of tumor vesicles (200 ug protein), reduced significantly thrombocytopenia and signs of respiratory sickness produced by the vesicles.

After these studies, activated Protein C, as well as nonact ivated Protein C, was given ip 15 min. and 3 hr after iv inoculation of 5×104 MCA mouse sarcoma tumor cells. Three weeks later mice were killed and lung tumors sized and counted. The data shown in the Table (average and range) indicate that activated Protein C decreases metastasis, while Protein C increases both the size and the number of tumors.

While the presumptive effect of activated Protein C on metastasis might be related to its anticoagulant and antiplatelet activities, we know nothing how Protein C enhances metastases in our experiments.
Inhibition Of Activated Protein C In Combined Factor V/VIII Deficiency
Authors: Giddings J C , Bloom A L
Published: July 2018

Human prothrombin complex concentrate was chromatographed on a dextran sulphate/sepharose column to provide fractions with the recognised properties of protein C. Fractions treated with thrombincoupled sepharose yielded samples which prolonged the activated partial thromboplastin time of normal human plasma and which had amidolytic activity against the chromogenic substrate S2238. A recent report indicated that normal human plasma inhibited this amidolytic activity whereas plasma from patients with hereditary combined factor V/VIII deficiency failed to do so (Marlar, R.A, and Griffin, J.H., J. Clin. Invest, 66: 1186, 1980). In the present study 21 plasma samples from patients of 11 different families with combined factor V/VIII defect were compared with normal plasma for their ability to inhibit the amidolytic action of thrombin- treated protein C. Six patients with classical severe haemophilia A and two patients with severe, isolated factor V deficiency were also studied. Plasma was mixed with activated protein C together with heparin and antikallikrein serum. Aliquots were removed at intervals and incubated with substrate S2238. Hydrolysis was detected in a spectrophotometer at 405 nm. In eight separate experiments normal plasma inhibited an average 63.5% (±15.6) of the amidolytic activity in 30 mins, incubation. Plasma from patients with haemophilia A or isolated factor V deficiency gave results which were not significantly different from normal (67.2% and 61% respectively). However plasma from patients with the combined defect inhibited an average of 24.5% (±13.6) of the amidolytic activity (p <0.01). Two of these plasma samples failed to inhibit any protein C activity. The results confirm that normal plasma contains an inhibitor of activated protein C and this appears to be deficient in patients with hereditary combined factor V/VIII defect.
Generation Of Factor XII-Dependent Plasminogen Activator Activity In Human Plasma Measured With A Fluorogenic Substrate
Authors: Dooijewaard G , Kluft C
Published: July 2018

A rapid fluorometric assay for measurement of amidolytic activity in human plasma was developed, using the plasminogen activator sensitive synthetic substrate t-BOC-L-valyl--glycyl-L-arginine-β-naphthylamide. The plasma is diluted in a reaction cuvet containing 0.050 M Tris HC1 buffer (pH 8.0) and 150 μM substrate. Activation of plasminogen proactivator(s) is initiated at 37°C by the addition of 10 μg dextran sulphate (MW 500,000)/ml. The concentration of β-naphthyl- amide released is recorded fluorometrically as a function of time. The slope of this curve at any time t is proportional to the concentration of activator. Thus, in a single assay, the entire time-dependent profile of activation and subsequent inhibition is monitored; this provides 1. a value for an optimum plasminogen activator content in the plasma, and 2. the time it takes to reach the optimum. The plot of optimum activator content against μl of plasma added is linear for dilutions more than 100-fold, suggesting that under these conditions the optimum content approaches the content of proactivator(s) originally present.

The activator content measured predominantly consists of contributions of a factor XII-dependent process since 1. without dextran sulphate or with plasmas deficient in factor XII or prekallikrein no activity could be generated, and 2. plots of optimum activator content against dextran sulphate concentration show sigmoidal-shaped saturation curves as found previously for the kallikrein generation in human plasma. Contributions of factor XIIa and kallikrein only partly account for the content measured and studies with plasmas deficient in factor XI point to a minor role for this factor, if any. Further identification of the activator (s) involved is in progress.
Fibrinolytic Properties Of Microbial Proteases
Authors: Egorov N S , Landau N S , Andreenko G V , Kreyer V G , Pokrovskaya S S , Karabasova M A , Lyutova L V
Published: July 2018

Our study of 820 microorganisms from various systematic and ecological groups revealed that 72% of the cultures contained enzymes caoable to dissolve human fibrin clots in vitro. Fibrinolytic enzymes are formed during growth of producers on strictly specific media. Changes in the composition of media and conditions of enzymatic synthesis were found to increase the specificity of microbial proteases to fibrin and to decrease their sensitivity to fibrinolytic enzymes inhibitors. A thrombolytic enzyme (TE) has been isolated and purified from the cultural fluid of actinomycetes. An intravenous injection of TE to albino rats results in an increase of fibrinolytic activity of the blood plasma euglobulin fraction. Time of euglobulin clot lysis is thereby decreased by 32%. The fibrinolytic activity measured from euglobulin precipitate on un-heated fibrin plates is increased 2.3-fold. However, the fibrinolytic activity of non-diluted blood plasma remains unchanged probably due to a sharp rise in antiplasmin content following TE injection. TE exerts marked thrombolytic effect in vivo. After intravenous injection of TE to animals with artificial clots in a v.jugularis segment a rapid clot lysis and reconstitution of blood flow are observed.
Anticoagulant And Fibrinolytic Properties Of The Salivary Gland Proteins From The Leech Haementeria Ghilianii
Authors: Budzynski Andrei Z , Olexa Stephanie A , Brizuela Barbara S , Sawyer Roy T , Stent Gunther S
Published: July 2018

Blood-sucking animals, such as leeches, mosquitoes, and vampire bats contain in their saliva agents that prevent the host blood from coagulating as it is being withdrawn. The work reported here was carried out to elucidate the nature of the anticoagulant found in the saliva of the giant leech Haementeria ghilianii, native to French Guyana. The two bilateral pairs of salivary glands of H. ghilianii are located at the proboscus base and encapsulated by a thin membrane. The anterior glands are 20 mm long and contain predominantly the cells of large size; the posterior glands are 5 mm long and contain the cells of very small size. Both salivary glands of the leech contain an anticoagulant that not only inhibits the clotting of human and bovine plasma, but also dissolves previously formed fibrin clots. This anticoagulant activity is attributable to an enzyme, for which the name hementin is proposed. Hementin catalyzes the proteolytic degradation of fibrinogen and fibrin, even in the presence of the inhibitors of proteases occurring in human plasma. The enzyme has the same affinity for human fibrinogen and fibrin. It cleaves human fibrinogen to yield characteristic fragments of high molecular weight that are different from Fragments X, Y, D and E resulting from the digestion of fibrinogen by plasmin. Both the anterior and posterior salivary glands contain hementin. Fractionation of salivary gland homogenates by differential ultracentrifugation showed that hementin is found entirely in the cytosol fraction. The salivary extracts do not contain any appreciable amounts of an activator of human plasminogen or an inhibitor of human or bovine thrombin, thus, Haementeria ghilianii prevents coagulation of its host’s blood through a fibrinogenolytic mechanism that is entirely different from that of hirudin, a thrombin inactivating polypeptide present in the saliva of another leech, Hirudo medicinalis.
Tolerancy Of Animal Fibrinolytic System To Exogenous Plasminogen Activator
Authors: Andreenko G V , Shimonaeva E E , Lyutova L V
Published: July 2018

Our previous studies showed that intravenous injection of tissue plasminogen activator (TPA) from pig heart to albino rats causes lysis of artificial clots in v.jugularis. This is concomitant with a short-term increase in the fibrinolytic activity (FA) of the euglobulin fraction of blood plasma. The other components of the fibrinolytic system remain thereby unchanged.

We tested the state of the fibrinolytic system at increasing concentrations of TPA and under varying conditions. A repeated (2-fold) injection of TPA did not further increase FA. When TPA was injected 4 times, the antiactivator and the level of TPA in the plasma of test and control animals decreased. This can be due to exhaustion of endogenous plasminogen activator as a stress response to repeated fixation of animals for intravenous injection of TPA. Injection of the same amount of TPA for 5 days caused no significant changes in FA. After a single injection of a 5-fold amount of TPA the changes were more noticeable; fibrinogen concentration was slightly decreased, whereas plasma recalcification time was increased. Thus, administration of exogenous TPA causing clot lysis has no prolonged effect on FA, which can be either due to its rapid release from the blood stream or to its binding to blood proteins.
The Preventive Effect Of The Polyunsaturated Fats On Thrombosis
Authors: Yotakis L D O
Published: July 2018

The fact of thrombosis, mainly the case of coronaritis, was examined in regard of a diet consisting of polyunsaturated fats by use of two dietary differing populations. The representative groups consisted respectively of (70) and (80) persons of the age-groups 45-55, selected by random process between males as generally females suffer a lower incidence of cardial thrombosis than men.

The first group concerned the universe of pure frugal meal, consisting of polyunsaturated fats, such as linon, linolen and arachidon acids having double bonds. The related population consisted of Gypsies and certain Coastal inhabitants the food of whom is obtained from vegetables, fishes and marine animals, containing the mentioned fats.

The second sample was selected between well-fed and gluttonous individuals, representing the population of frequent coronaritis and thrombotic myocardium infarctus.

42 attacks of thrombosis were observed among the 80 persons of the so-called well-fed sample; consequently the incidence-rate was 52 per cent. On the other hand the reciprocal rate concerning the frugal diet was equal to 3 per cent, as only 2 cases of attack were observed between Gypsies and the Coastal inhabitants. The rate of the two groups together was 29%. All other factors such as psychic stress, etc., being equal, the difference in thrombosis between the two populations 52 - 3 = 49 per cent is significant, as by means of the standard error of difference equal to 7 we observed that it is more than might occur by chance. The results of the investigations suggested that the polyunsaturated fats provoking and converting certain antiaggregating bodies and the vasoactive prostaglandins as well as a representative diet are efficient to the prevention of thrombosis.
Role Of Platelet cAMP And Prostaglandin Synthesis In Platelet Aggregation Inhibition By Ticlopidine Hydrochloride
Authors: Bruno J J , Yang D , Taylor L A , Feamster C
Published: July 2018

Ticlopidine hydrochloride (T), 5-[(2-chlorophenyl) methyl]-4,5,6,7-tetrahydrothieno [3,2-c] pyridine hydrochloride, is a potent antiplatelet agent of unknown mechanism of action. T does not inhibit human platelet low Km, cAMP phosphodiesterase (PDE) in vitro, (IC50>>10-3M), but does have some activity versus the low Km, cGMP-PDE (IC503-4×10-4). Equivalent values for theophylline, a weak PDE inhibitor, are 1.6×10-4M and 4.2×10-4M, respectively. T has no synergistic effect in vitro on platelet aggregation inhibited by PGE1. PDE inhibitors do show synergism with PGE1 and other antiaggregatory PGs. Ex vivo in humans and animals, T inhibits ADP-induced aggregation. This inhibition is not altered by addition of SQ-22536 (f.c.-10-4M), a purported inhibitor of adenylate cyclase activity, whereas drugs whose platelet inhibitory activity depend on elevation of platelet cAMP, e.g., PGE1, adenosine and dipyridamole, have this inhibition partially reversed by SQ-22536.

PG synthesis is not required for the antiplatelet activity of T. Addition of aspirin (f.c.-5×10-4M) in vitro to PRP from humans treated with T (500 mg/day) did not modify the aggregation response to ADP. In addition, PGI2 is not essential for the antiaggregation effect of T.

The inhibition of ADP-induced aggregation of PRP prepared from blood of T-treated humans just after collection and more than 30 minutes after, to allow for inactivation of PGI2 were identical.

We conclude that neither elevated cAMP nor PG synthesis is essential for T activity.
Potentiation Of Anti-Aggregating Prostaglandins By Ticlopidin
Authors: Bonne C , Martin B , Regnault F
Published: July 2018

Ticlopidin (T) is a potent inhibitor of platelet aggregation which enhanced the platelet sensitivity to anti-aggregating prostaglandins such as PGE1. The goal of this study was to examine the site of action of T in the rat platelets after in vivo administration, in respect to the PGE1 mechanism of action.

Male adult Wistar rats were orally administered at the dose of 200 mg/kg, 20 and h hours before blood collection. The binding of (3H)-PGE1 to platelets from controls and treated rats was determined by filtration technique. The c AMP formation was measured by protein binding assay in aspirin-washed platelet suspension or in lysate after stimulation by PGE1, adenosine or NaF.

Since potentiation of PGE1 by T occured in aspirin-washed platelets, it is independent of the ability of the cells to synthesise prostaglandins. On the other hand, PGE1-receptor binding is not affected by T treatment and potentiation of PGE1 effect is not specific. Moreover, basal adenylate cyclase activity is increased in platelet particulate fraction of treated rats, but NaF stimulation is not enhanced.

These results suggest that T activates the adenylate cyclase systems by an action on the coupler subunit of the enzyme just as guanosine triphosphate does.
Antiplatelet Effect Of A New Compound, Imidazo[1,2-a]- Thienopyrimidin-2-One : A Cyclic AMP Mediated Phenomenon
Authors: Ashida S , Sakuma K , Abiko Y
Published: July 2018

The effect of a new compound, 1,2,3,5,6,7,8,9-Octahydro- [1]benzothieno[2,3-d]imidazo[1,2-a]pyrimidin-2-one hydrochloride (DH-6471), on cAMP metabolism and aggregation of platelets was studied. In vitro. DH-6471 inhibited platelet aggregation (both the 1st and 2nd phases) induced by ADP, collagen, thrombin, arachidonic acid and PGG2-TXA2 mixture in PRP from various animal species including human at concentrations (IC50) ranging from 0.07 to 8 μH. It inhibited ADP- and collagen-induced platelet aggregation ex vivg in rats following oral doses as low as 0.3- 1 mg/kg.

The compound was found to be a highly selective inhibitor of platelet low Km cAMP phosphodiesterase (Ki=0.025 μM), when tested with enzyme fractions separated by DEAE-cellu-lose chromatography. It did not significantly affect basal or PGE1(0.1-1 μM)-stimulated cAMP level of platelets at a concentration of 1 μM where platelet aggregation and the low Km PDE were markedly inhibited. However, both basal and PGE1-stimulated accumulations of cAMP in the platelet membrane fraction were increased by DH-6471 at 1 μM when the isolated membrane fraction was incubated with ATP-Mg2+.

Studies with several PDE inhibitors including papaverine, dipyridamole and DH-6471-related compounds showed a close correlation between their ability to inhibit the low Km PDE or to increase cAMP accumulation in the membrane fraction and their inhibitory effect on platelet aggregation. On the other hand, their potency to inhibit high Km cAMP-PDE(cGMP-PDE) and to increase cAMP level in whole platelets was poorly correlated to their inhibitory activity in platelet aggregation.

These results suggest that some small but local changes in platelet cAMP may be involved in the regulation of platelet aggregation, particularly primary aggregation.
Deposition Of Fibrin, Fibrinogen And Platelets On The Sub- Endothelium Of Ex Vivo Perfused Rabbit Aorta
Authors: Neftel K , Baumgartner H R , Haeberli A , Straub P W
Published: July 2018

The subendothelium of 2 cm segments of rabbit aorta was exposed to human blood flowing directly from an antecubital vein under controlled conditions, the flow being 10 and 20 ml/min., the perfusion time varying between 1 and 10 min.. One cm of aorta taken from the middle of the segment was incubated with plasmin until proteolysis of fibrin and fibrinogen was completed. From quantitative measurements of the proteolytic fragment E, the sum of fibrinogen + fibrin was calculated (J.Lab. Clin. Med. 96:258,1980). The proteo- lysate was then incubated with thrombin, the released fi-brinopeptide A determined using a RIA and the fibrinogen of the deposits calculated. In addition, fibrin and platelets deposition were estimated morphometrically on the adjacent distal and proximal part of the segments.

1. Fibrin deposits varied between 1.4 and 82.6 ug. At a flow of 10 ml/min. fibrin deposition increased7 with increasing perfusion time; at 20 ml/min. no time dependence was observed.
2. Platelet adhesion, deposited platelet volume and thrombus height increased with time at both flew rates.
3. Fibrinogen varied between 1.4 and 7 ug. In contrast to fibrin maximal levels were reached witnin one minute.

Thus, in the ex vivo system fibrin deposition was dependent on flow and with the lower flow rate also on perfusion time. In contrast, the observed fibrinogen was probably due to unspecific adsorption.
Thrombin-Induced Release Of Adenine Nucleotides From Cultured Human Endothelial Cells
Authors: Lollar P , Owen W G
Published: July 2018

The effect of thrombin on the release of adenine nucleotides from cultured human umbilical vein endothelium was studied. When endothelial cell monolayers were incubated with 3H-adenosine for two hours at 37°C, fifty percent of the radioactivity was incorporated into the cell. Thin-layer radiochromatography revealed that greater than 90% of the tritium was associated with ADP and ATP.

Addition of thrombin to the monolayers resulted in the release of tritium into the medium. This effect was dose-dependent and saturable, with a maximal response seen at 1 × 10-8 M thrombin. Diisopropylphosphoryl thrombin (DIP-thrombin) , which is enzymatically inactive, did not cause a release of tritium. Blocking the high-affinity thrombin binding sites which are present on the endothelial cell surface was done with DIP-thrombin. Saturating these binding sites by greater than 95% with a fifty-fold excess of DIP-thrombin had no effect on thrombin-induced release, indicating that the high-affinity binding sites are not involved in the release of adenine nucleotides by thrombin.
Effect Of Thrombin On Platelet Adherence To Cultured Vascular Cells: Modification By Dapa
Authors: Czervionke R L , Hoak J C , Haycraft D L , Fry G L
Published: July 2018

The role of thrombin in inducing adherence of platelets to cultured vascular cells is complex and may involve factors of both platelet and vascular origin. Dansylarginine N- (3-ethyl-l,5-pentanediyl)amide, DAPA, a fast-acting inhibitor of thrombin, was used in this study to evaluate platelet activation and endothelial PGI2 release induced by thrombin. Monolayers of endothelial cells and fibroblasts were cultured from human umbilical cord vessels. Empty culture dishes were used as a control. Some vascular cells were treated with aspirin (ASA) to inhibit PGI2 formation before they were incubated with 51Cr-platelets for platelet adherence studies. PGI2 was assayed by radioimmunoassay for 6-keto-PGF1α. In one study, endothelium was incubated with 0.3 U thrombin for 2 min. before 10 μM DAPA was added. Platelets were next mixed with this solution and adherence was determined. DAPA caused a decrease in thrombin-induced platelet adherence to normal endothelium from 8% to 2%. When ASA- treated endothelium was employed, DAPA decreased adherence from 59% to 2%. In another study, 51cr-platelets were first aggregated with 0.1 U thrombin, then 1.3 μM DAPA was added, and these aggregates were incubated with the monolayers. Similar experiments without DAPA served as positive controls. DAPA did not reverse the aggregate formation, but it did reduce platelet adherence to normal endothelium from 38% to 1%, and to ASA-treated endothelium from 68% to 11%. In contrast, DAPA had little effect with fibroblasts (81% to 71%), ASA-treated fibroblasts (82% to 67%), and the empty dish control (86% to 66%). These results suggest that thrombin-induced platelet adherence in this system involves more than just an effect upon platelets. In addition, they provide further evidence that the non-thrombogenic nature of endothelium persists despite the absence of PGI2.
Postpartum Cytotoxic Endothelial Antibodies
Authors: Parsons T , Hoak J , Schroeder C , Goeken N , Thompson J
Published: July 2018

HLA and non-HLA antigens have been identified on human endothelial cells (HEC). A double fluorochromatic assay was used to detect specific and nonspecific cytotoxic antibodies to endothelial antigens in sera from postpartum women and in patients with thrombotic disease. Single donor umbilical vein HEC were placed in histocompatibility typing wells, and incubated overnight at 37°C, 4% CO2 atmosphere, to form a monolayer. Five μl of test serum was added to each monolayer and incubated for 1 hr. at 37°C. Serum was removed and five yl non-toxic rabbit complement was incubated with the HEC for 2 hrs.at 37°C. Staining was then performed with di- acetylfluorescein and ethidium bromide, and the extent of cytotoxicity was expressed as per cent cell kill. Fifty-six consecutive consenting mothers had 72 hour postpartum sera screened. Cytotoxic antibody to endothelium was present in 10. Three of the 10 sera scored greater than 80 per cent kill with over 80 per cent of the HEC monolayers. Four other postpartum sera that had exhibited HEC cytotoxicity were studied. These 7 sera shared additional characteristics: (1) all showed 60 to 100% reactivity with standard lym- phocytotoxicity testing, (2) all failed to induce platelet aggregation, and (3) complement-mediated HEC toxicity was removed by adsorption of the sera with pooled platelets in 6 of 7. The clinical relevance of this cytotoxic HEC activity has not been delineated but an association was found in a 28 yr. old, twice pregnant woman with recurrent deep vein thrombosis and pulmonary embolism. Her anti-endothelial antibody characteristics were like those found in the 7 postpartum women. These results suggest a potential relationship between endothelial injury and thromboembolic complications.
Viral Replication And Platelet Adhesiveness To Vascular Endothelium
Authors: Chernesky M , Bromke A , Frame B , Larke B , Galet H , Prevec L
Published: July 2018

Newcastle disease virus (NDV) and chikungunya virus (CV) replicated in monolayers of human umbilical vein endothelium to peak titres of 104.5 TCD50 per ml in 24 and 48 hours respectively following inoculation of 106 TCD50/ml of virus. The cultures demonstrated no cytopathic effects after 6 days incubation. Analysis of intracellular protein from virus-infected endothelial cells by polyacrylamide gel electrophoresis of 35s-methionine labelled extracts of infected and uninfected cells indicated that at 22 hours after infection, normal cellular protein synthesis was shut down and virus-specific protein was found inside the cells. In contrast, Influenza A viruses (HK and PR 8) did not replicate in endothelial cells. Endothelial cells exposed to a high dose of NDV were severely damaged as illustrated by rounding and stranding. Influenza A and CV did not exhibit this toxic effect. Although both NDV and CV demonstrated replication, they showed contrasting thrombo- genic effects in virus endothelium platelet reactions. Larger amounts of radioactivity were associated with endothelial cells when washed suspensions of 51Cr-labelled human platelets were added 24 hours after infection with NDV than when exposed to heat inactivated NDV or allantoic fluid. Platelets added to cultures infected with non-toxic doses of NDV or influenza virus adhered singly and in clumps to the surface of the endothelial cells, but not to those infected with CV. Identical results were attained when platelets, pre-incubated with virus, were placed on uninfected endothelial cell monolayers, then washed with buffer. The mechanism by which this adhesion takes place may be due to virus bridging or modification of endothelial cell membranes by viral neuraminidase leading to a depression of endothelial cell properties responsible for antiadhesiveness.
Structural Studies On Glycocalicin
Authors: Jamieson G A
Published: July 2018

When platelets are homogenized, a major membrane glycoprotein component (Mr 150,000) which has been termed glycocalicin, appears in the supernatant in soluble form. Immunological studies in several laboratories suggest that glycocalicin is derived from glycoprotein Ib (Mr 200,000) by the action of a Ca++ - dependent protease released from the platelet.

Glycocalicin has been purified to homogeneity by conventional procedures as well as by a two step procedure utilizing affinity chromatography on insolubilized wheat germ agglutinin and insolubilized thrombin. Purified glycocalicin has been shown to inhibit platelet aggregation induced by thrombin and by ristocetin.

The molecule is cleaved by trypsin to a macroglyco- peptide (Mr 120,000) in which biological activity has not been detected, and a peptide “tail” (Mr 45,000), which is a competitive inhibitor of the binding of thrombin to intact platelets.

Intact glycocalicin contains ∼ 60% carbohydrate comprising Gal, GalNAc, GluNAc and NeuNAc in the ratio 2:1:1:2. The macroglycopeptide contains virtually all of this carbohydrate. Only trace amounts of carbohydrate are found in the tail peptide but serine, glycine and glutamic acid comprise over half the amino acid residues. Glycocalicin in intact platelets is resistant to iodi- nation but is readily iodinated following isolation both in the macroglycopeptide and tail peptide portions of the molecule.

The ready availability of purified glycocalicin may be of value in elucidating structure-function relationships of glycoprotein Ib and its orientation in the platelet membrane.
Production Of PGI2 By Cultured Endothelial Cells And Smooth Muscle Cells
Authors: Jaffe E A
Published: July 2018

Prostacyclin (PGI2) is the main product of the cyclooxygenase pathway in endothelial cells and is a short-lived vasodilator and inhibitor of platelet aggregation. Cultured endothelial cells (EC) make PGI2 from exogenous arachidonic acid (AA) and the unstable endoperoxide PGH2. EC have also been shown to make PGI2 from endogenous AA when stimulated by the calcium ionophore A23187, thrombin, histamine, bradykinin, trypsin, platelet derived growth factor, and uremic plasma. PGI2 synthesis in EC is blocked by aspirin, indomethacin, or 15-HPAA. PGI2 synthesis is partially self-controlled and self-limiting at least in vitro since PGI2 elevates EC cAMP levels and elevated cAMP levels decrease EC PGI2 production. While the proteolytic site of thrombin (and trypsin) is involved in the stimulation of PGI2 production, high affinity binding of thrombin to endothelial cells appears not to be necessary. Alteration of the fatty acid composition of EC by manipulation of the culture media can decrease PGI2 production in response to thrombin or A23187 by 60-751. Cultured aortic smooth muscle cells (SMC) also synthesize PGI2 but at significantly lower levels than EC. SMC PGI2 production is stimulated by PDGF and in some SMC lines by thrombin. SMC grown from atherosclerotic rabbit aortas produce considerably less PGI2 from exogenous AA than do SMC from noimal aortas. Future investigations into this area will further clarify normal biochemical and physiologic pathways and perhaps suggest means by which PGI2 production in EC and SMC may be modulated in order to treat human disease.
Structure-Function Relationships Of Heparin
Authors: Rosenberg R D , Oosta G M
Published: July 2018

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa- antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin-antithrombin complex formed within the reaction mixture. Plots of the initial velocity of the factor IXa-anti-thrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin-antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. Thus, our data demonstrate that the binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of these four enzymes. In the case of factor Xa and plasmin, the heparin induced acceleration of these two processes appears to be due to the direct interaction of mucopolysaccharide with protease inhibitor. Structural analyses of small heparin fragments suggest that the above biologic action of the mucopolysaccharide is dependent upon a protease inhibitor binding domain which contains two non-sulfated uronic acid residues. In the case of thrombin and factor IXa, the heparin induced enhancement of enzyme neutralization appears to be secondary to the direct binding of heparin to antithrombin as well as upon interactions between free enzyme and complex carbohydrate attached to inhibitor (approximation effect). Structural analyses of larger heparin fragments indicate that these direct and approximation actions of the mucopolysaccharide are dependent upon multiple discrete sites which lie on the non-reducing side of the protease inhibitor binding domain.
Antithrombin - Mechanism Of Action And Binding Of Heparin
Authors: Björk I , Lindahl U
Published: July 2018

Antithrombin inhibits a variety of serine proteases by forming equimolar, inactive complexes with the enzymes. The anti thrombin-thrombin complex, extensively studied as a model for complexes with other coagulation proteases, dissociates with a half-life of several days to free enzyme and a proteolytically modified inhibitor. It thus behaves like a kinetically stable enzyme-substrate complex. Several observations indicate that deacylation is the rate-limiting step. The active site of antithrombin, i.e. the bond slowly cleaved by the target enzyme, is the Arg-385/Ser-386 bond in the carboxy-terminal region of the protein. The formation of most anti thrombin-protease complexes is greatly accelerated by certain forms of heparin. These active molecules comprise about 1/3 of normal heparin preparations and bind with high affinity (K∼108 M-1) to the inhibitor, regardless of the size of the polysaccharide. The stoichiometry of binding is 1:1 for most heparin molecules, although some high-molecular-weight chains have two antithrombin binding sites. Evidence from spectroscopic and kinetic analyses suggests that the binding of high-affinity heparin induces a conformational change in antithrombin that probably is involved in the mechanism of the increased rate of enzyme inactivation. Oligosaccharides with high-affinity for anti thrombin have been isolated by affinity chromatography following partial deaminative cleavage of heparin with nitrous acid. The smallest such oligosaccharide obtained is an octasaccharide, in which a pentasaccharide sequence appears to comprize the actual antithrombin-binding site. This active sequence contains a unique, 3-O-sulfated glucosamine residue that does not appear to occur in other portions of the heparin molecule. In addition, two N-sulfate groups and probably at least one O-sulfate group within the pentasaccharide sequence are essential for high-affinity binding of heparin to antithrombin.
The Disulfide Bond In Antithrombin Required For Interaction With Heparin
Authors: Finlay T H , Ferguson W S
Published: July 2018

Mild reduction of antithrombin (AT) with dithiothreitol followed by alkylation with [14C]-iodoacetamide results in the incorporation of 1.7 mol of acetamide/mol of protein with cysteine the only amino acid modified. Tryptic digestion of the reduced and alkylated AT results in the formation of only two labeled peptides indicating that mild reduction cleaves only one of the 3 disulfide bridges.

The 2nd order rate constant for the reaction of thrombin with both native and mildly reduced AT is 5.9-9.6 × 105 M-1 min-1. Addition of heparin accelerates the reaction between native AT and thrombin (k >107 M-1 min-1) but has no effect on the reaction between reduced AT and thrombin (k = 8.3-12.2 ×105 M-1 min-1). Alkylation of reduced AT destroys all remaining thrombin-neutralizing activity.

Native AT elutes from a heparin-Sepharose column with 1.0 M NaCl whereas reduced AT elutes at 0.5 M NaCl. Reduced and alkylated AT is not retained by the immobilized heparin even in 0.1 M NaCl. The intrinsic tryptophan fluorescence enhancement observed following binding of heparin to native AT is not seen with reduced AT. These data indicate that reduction of a single disulfide bond in AT lowers the affinity of the protein for heparin and eliminates the heparin-mediated acceleration of thrombin neutralization.

Mildly reduced and alkylated AT was digested with CNBr and the peptides were fractionated by HPLC. Preliminary analysis of the CNBr peptides suggests that the crucial disulfide bond is between CYS239 and CYS422 (using the numbering in Magnusson’s sequence data).

Supported by grants from the USPHS. THF is a Research Career Development Awardee of the NIH (HL-00277).
Effect Of Thrombin On The Production Of Plasminogen Activator By Endothelial Cells
Authors: Loskutoff D J
Published: July 2018

Cultured bovine aortic endothelial cells (ECs) were employed to determine whether agents associated with the forming thrombus alter the fibrinolytic activity of the endothelium. The addition of thrombin to the cultures caused a rapid decrease in intracellular plasminogen activator activity (PAi) as measured by the plasminogen-dependent hydrolysis of radiolabeled fibrin. The cells were extremely sensitive to thrombin with as little as 3 ng/ml causing a 50% decrease in PAi within 30 min. This decrease 1) occurred in cells pretreated with actinomycin D, cycloheximide or colchicine, 2) was relatively specific since no decrease was initiated with trypsin, pronase, collagenase, or chymotrypsin, and 3) did not appear to result either from secretion or by direct inactivation of PA by thrombin. Thrombin inactivated with diisopropylfluorophosphate did not elicit the response. PAi returned to control levels within 6 hr after the addition of the thrombin inhibitor, hirudin. This reversal by hirudin was blocked by cycloheximide but not by actinomycin D. The decrease in PAi correlated with the disappearance of a form of PA of Mr 48,000. Other forms produced by the cells (i.e., 100,000, 74,000 and 53,000) were unaffected as revealed by polyacrylamide gel electrophoresis in the presence of SDS. We have begun to characterize the effect of thrombin on other metabolic activities of ECs. It was not mitogenic for growing or confluent cultures, did not alter the rate of RNA or protein synthesis, did not effect the release of 51 chromium from the cells, and apparently did not alter the spectrum of proteins synthesized by the cells. These results indicate that suppression of PAi activity by thrombin is dramatic, rapid, reversible and highly specific, and may be effected at the level of the actual synthesis of the molecule. The generation of thrombin at sites of vascular injury may thus protect the growing thrombus against premature, EC-mediated dissolution.
Multiple Molecular Forms Of Plasminogen Activator Produced By Cultured Bovine Endothelial Cells
Authors: Levin Eugene G , Loskutoff David J
Published: July 2018

The production of plasminogen activator (PA) by the vascular endothelium has been implicated in the maintenance of vessel patency. Cultured bovine aortic endothelial (BAE) cells were employed to study and compare the cell associated and secreted forms of PA. Samples were fractionated by polyacrylamide gel electrophoresis in the presence of SDS. PA activity in the gel was localized by the fibrin-overlay technique. Cellular PAs were found to be membrane associated and to consist of a major form of Mr 48,000 (C48) and minor forms of 53,000 (C53), 74,000 (C74), and 100,000 (C100). Incubation of the cell extracts at 37°C resulted in the appearance of two additional forms of Mr 41,000 and 33,000 suggesting that these forms were degradation products. Serum-free conditioned medium (CM) contained secreted PAs of Mr 52,000 (S52), 55,000 (S55), 74,000 (S74) and 100,000 (S100). In addition, a broad zone of fibrinolytic activity was observed in the region between Mr 80,000 and 95,000. Cellular PAs have isoelectric points of pH 8.5-8.6 and 7.5 while secreted PAs demonstrate activity at pH 8.6, 8.5, 8.0, and 7.5. The forms showed differential sensitivities to DFP with S74 and C74 being inactivated by 1mM DFP within one Hr at 37°C while S52 and C48 were still partially active after treatment with 10mM for two Hrs. S100 was completely retractile to treatment with 40 mM DFP. The addition of fresh serum to confluent cultures resulted in the disappearance of C48 and C74, and of S52 and S74, but caused an increase in C100 and S100. These studies indicate that several forms of PA are produced by endothelial cells and suggest that the production of each may be independently regulated.
Inhibition Of Collagen And ADP-Induced Platelet Aggregation By Plasma Fibronectin
Authors: Moon D G , Kaplan J E
Published: July 2018

Platelets contain fibronectin a glycoprotein with an established affinity for collagen. This observation has led other investigators to postulate that fibronectin is the platelet collagen receptor. The much greater concentration of fibronectin in the plasma surrounding platelets, however, has led us to suggest that plasma fibronectin may bind to collagen and competitively inhibit the platelet- collagen interaction. Rat platelets were isolated by Stractan density gradient centrifugation and aggregated with acid-solubilized rat tail tendon collagen (Type I) in a Payton 300B Aggregometer. Fibronectin was twice purified by affinity chromatography with gelatin linked to CNBr- activated Sepharose 4B. Simultaneous addition of 50 μg fibronectin and 25 μg collagen to platelets suspended in Tyrodes solution at 37°C resulted in a 2-fold increase in lag time and a 30% decrease in aggregation rate as compared to control values. When collagen was preincubated in Tyrodes solution for 12 minutes at 26°C without platelets to allow for prior fibrillogenesis, the addition of 50 μg fibronectin with the platelets resulted in <20% increase in lag time and a 20-30% decrease in aggregation rate. In a separate series of experiments, fibronectin was also found to inhibit ADP-induced aggregation. In this case, the initial rate of aggregation was comparable with and without fibronectin, but this maximal rate was maintained for a shorter period in the presence of fibronectin. Thus, fibronectin reduced the in vitro aggregation response to two different physiological stimuli. Our data supports previous studies which indicate that fibronectin reduces the reactivity of platelets with collagen and provides evidence of a role for fibronectin in modulating platelet responses in the absence of collagen.
Thrombasthenic Platelets Do Not Bind Plasma Fibronectin
Authors: Ginsberg Mark , Chediak Juan , Lightsey Alton , Plow Edward F
Published: July 2018

Plasma fibronectin (fn) binds to thrombin-stimulated normal platelets and may thus contribute to platelet participation in hemostasis. Glanzmann’s thrombasthenia is an inherited bleeding disorder in which the affected platelets do not aggregate in response to thrombin or other stimuli and have diminished ability to retract clots. To evaluate the ability of thrombasthenic platelets to bind fn, suspensions of gel filtered platelets and 2nM 125I fn were incubated at 37°C in the presence or absence of 2 units/ml purified human thrombin. At various times, cells were centrifuged through 20% sucrose in microfuge tubes and bound 125I fn measured in amputated tube tip. In the case of normal cells, in the presence of thrombin, there was time dependent uptake, plateauing by 20 min incubation. At equilibrium, greater than 95% of binding was saturable, and Scatchard plots indicated a single class of binding sites with Kd = 3×l0-7 M and a maximum of 120,000 molecules/platelet. No binding was observed in the absence of thrombin. In contrast, platelets from 7 thrombasthenic individuals from 4 unrelated kindreds showed little or no detectable uptake of fn in the presence or absence of thrombin even after a 60 min incubation. Nevertheless, thrombin did induce serotonin secretion from these cells, showing that stimulation was occurring. Thrombasthenic platelets are defective in their ability to bind fibrinogen in the presence of ADP. These data indicate a defect in platelet interaction with another plasma protein in this disease. They also suggest the possibility that deficient fn-platelet interaction contributes to the thrombasthenic bleeding diathesis.
Fibronectin-Binding Glycoprotein From Human Platelet Membranes
Authors: Hansen M Sandbjerg , Clemmensen Inge
Published: July 2018

Fibronectin (cold insoluble globulin) has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein was partially purified from washed solubilized human plate letmembranes by affinity chromatography on fi- bronectin-Sepharose. The isolated protein migrated as a single band in SDS-polyacrylamidegelelectro- phoresis with a molecular weight of approximately 125000 under reducing conditions. In non-reduced gels the protein migrated as a dimer. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmuno assay. The protein and purified fibronectin formed a complex which had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding proteinfibronectin mixture resulted in an even faster mobility of the complex, while the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetyl-glu- cosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin binding glycoprotein in the platelet membrane.
A Study On Fibrinolysis Induced By Defibrase Infusion
Authors: Kato M , Fujimaki M , Fukutake K
Published: July 2018

When Defibrase as a defibrination agent was infused intravenously, it was followed by fibrinolytic phenomenon with release of FDP in high concentration. In this paper it will be reported which in actual inducer among fibrino- peptide A, des-A-fibrinmonomer and Defibrase itself in this occasion.

The activity of plasminogen tissue activator, plasminogen and α2 plasmin inhibitor were measured by chromogenic method using S-2322, S-2251 and S-2251 respectively and fibrinogen concentration was assayed by tyrosine method. FDP, plasminogen and α2 plasmin inhibitor were also measured by immunological methods.

After the infusion of Defibrase into four normal adults fibrionlytic activities were detected on the plasminogen-free fibrin plate and followed by decrease of plasminogen and α2 plasmin inhibitor and increase of FDP with plasminogen activator. On the other hand, Defibrase did not show any activities of plasminogen activator and plasmin. Therefore, it might be imagined that one of three substances including fibrinopeptide A, des-A-fibrinmonomer and Defibrase might be actual inducer of plasminogen activator from the vessel wall. When fibrinopeptide A separated from dog plasma was infused intravenously to dogs, no effect on fibrinolytic phenomenon, was indicated and, when the test-samples from the reflux experiments with Defibrase by the ues of dog’s leg were applied on standard fibrin plate, weak fibrinolytic reaction were detected in a few cases. However, when des-A-fibrinmonomer was tested, increase of FDP, decrease of fibrinogen and α2 plasmin inhibitor, showing the mode of dose-response, were detected in all cases of dog.

According to the results obtained above, it might be suggested that the actual inducer of fibrinolytic related with Defibrase infusion might be des-A-fibrinmonomer.
Plasminogen Activators In Human Malignant Melanoma
Authors: Markus G , Madeja J , Evers J L , Hobika G H , Camiolo S M , Ambrus J L
Published: July 2018

The plasminogen activator (PA) content of metastatic malignant melanoma was determined in Triton X-100 extracts of 11 surgical specimens and adjacent normal tissue, using azocaseinolysis with added plasminogen. The mean PA content of the tumors was 8.4 ± 10 (SD) CTA u/g tissue (6 times that of the surrounding tissue), lower than found earlier in lung, colon and breast tumors. Inhibition by goat IgG against purified human urokinase showed that the predominant activator was of the UK type, as was the case with the tumors examined earlier, except those of the prostate. This is in contrast with recent reports which showed that human melanoma-derived cells secrete into the culture fluid almost exclusively the “vascular type” PA (Wilson et al., Cancer Res. 40, 933, 1980; Roblin and Young, Ibid. 40, 2706, 1980). While this type of PA was present in all extracts here examined, in 3 of them only trace amounts (<1%) could be found. The vascular type PA, when present, could be inhibited by rabbit IgG against human melanoma cell culture-derived vascular PA (kind gift of Dr. E. Dowdle). SDS-gel electrophoresis in conjunction with fibrin-agar overlay zymography showed multiple activator bands ranging in mol. wt. from 100,000 to 30,000. All but the 70,000 mol. wt. vascular type PA band were inhibited by inclusion of anti-UK IgG into the fibrin-agar mixture.

The discrepancy between the tumor extracts and the culture media may be due to a selective advantage of vascular type PA-secreting cells in culture, or to the turning off of the UK gene in the culture environment. It is also possible that production of vascular type PA is suppressed iii vivo.