bluuebunny/crossref_metadata_2025 huggingface dataset:
ADRENALINE ACTIVATES HUMAN PLATELETS BUT IS NOT PER SE AN AGGREGATING AGENT. EFFECTS ON PLATELET MORPHOLOGY, MEMBRANEFLUIDITY, FIBRINOGEN BINDING, CYTOPLASMIC FREE CALCIUM AND PROTEIN PHOSPHORYLATION
Authors: Lanza M , Beretz A , Stierlé A , Hanau D , Kubina M , Cazenave J P
Published: August 2018

Adrenaline (Adr) is generally considered as a full agonist able to induce in vitro the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. Adr 2.5 M) induces the aggregation and secretion of 41 % of preloaded 3H-serotonin in human platelets in citrated plasma. This effect is not seen in plasma collected on 50 ATU/ml hirudin, and is due to the generation of traces of thrombin during blood collection and not to a direct effect of citrate itself, such asthe lowering of plasma free calcium. With washed human platelets suspended in Tyrode's buffer containing 2 mM Ca2+, 0.35 %albumin and apyrase, Adr (0.1 -100 M) doesnot cause shape change, aggregation or secretion of serotonin and does not modify platelet ultrastructure as judged by electron microscopy. Adr (1-100 M) does not change platelet membrane fluidity, as studied with the lipophilic fluorescent probe TMA-DPH. Adr has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+levels measured with quin2, or phosphorylation of 20 KDa or 47 KDapolypeptides, whereas all these parameters are modified after stimulation with ADP orthrombin. Adr potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels,membrane fluidity, fibrinogen binding or protein phosphorylation. This effect is also seen with alpha2-adrenergic agonists (noradrenaline, alpha-methyl noradrenaline, clonidine) and is inhibited by alpha2-adrenergicantagonists such as yohimbine. The potentiation of platelet aggregation by Adr is not modified by prior incubation of the platelets with1mM aspirin for 15 min. This study shows that Adr alone does not induce modifications ofmorphology, metabolism or function of intactand functional washed human plateletsand that Adr cannot be considered per se as an aggregating agent. However, Adr interactswith alpha2-adrenergic receptors on human plateletsand potentiates biochemical and aggregatory responses induced by other platelet agonists.
TISSUE PLASMINOGEN ACTIVATOR INHIBITS THROMBIN-INDUCED AGGREGATION AND SHAPE CHANGE,BUT FACILITATES SECRETION,IN GEL-FILTERED PLATELETS ONLY
Authors: Blockmans D , Houtte E Van , Arnout J , Mombaerts P , Collen D , Vermylen J
Published: August 2018

Prolonged administration of tissue plasminogen activator (t-PA) has caused bleeding problems in some patients, that did not necessarily correlate with a significant drop of fibrinogen levels. We have therefore evaluated the effect of t-PA on platelet function in vitro.

Incubation of gel-filtered platelets for one hour at 37°C with 180 μg/ml plasminogen and increasing concentrations of t-P(50-1600 ng/ml) significantly inhibited shapechange and aggregation induced by thrombinand the thromboxane mimetic U 46619 in a dose-dependent manner. In an EDTA milieu, whichbolishes aggregation, a dual effect of t-PA and plasminogen was observed in the aggregometer: the thrombin- or U 46619-elicited initial decrease in light transmission, reflectingthe disc-to-sphere transformation of platelets, was almost completely inhibited from 50 ng/ml t-PA upwards; the subsequent increase in light transmission, reflecting granule secretion, was however enhanced by small amounts of t-PA (up to 200 ng/ml). The latter finding was confirmed by direct measurement of secreted ATP:t-PA atconcentrations up to 200ng/ml enhanced thrombin- or U 46619-in-duced secretion. The amount of plasmin generated in the gel-filtered platelets-plasminogen-t-PA mixtures was quantified. The same amountsof plasmin, while also inhibiting the disc-to-sphere transformation of the platelets, did not enhance thrombin-or U 46619-induced ATP secretion. When whole blood or platelet-rich plasma or gel-filtered platelets resuspended in α-antiplas-min-depleted plasma waspreincubated with t-PA, aggregation and/or shape change induced by ristocetin, arachidonic acid, the calcium ionophore A 23187, adenosine diphosphate, U 46619, thrombin, serotonin or platelet activating factor were not modified.

Our results suggest that in a purified system the effects of t-PA plus plasminogen onplatelets are distinct from those of plasmin;it appears that low pharmacological concentrations of t-PA enhance the secretory responses to stimuli.
PLATELET FUNCTION IN PLASMA AT PHYSIOLOGICAL CALCIUM CONCENTRATION. THE USE OF VAC AS AN ANTICOAGULANT
Authors: Gool R Van , Reutelingsperger C P M , Hornstra G , Hemkera H C
Published: August 2018

We have purified from human placenta an anticoagulatory protein (VAC, Mr= 32,000), which inhibits phospholipid dependent procoagulant reactions through a calcium dependent high affinity binding to procoagulant phospholipids..When tested in citrated platelet rich plasma (cPRP), VAC does not affect platelet aggregation and secretion in reponse to ADP, collagen and thrombin. Purified VAC (0.05 μM, final concentration; f.c.) was used as an anticoagulant to prepare PRP (VAC-PRP). Platelet aggregation (optical density method) and release of newly absorbed 14C-sero-tonin (5HT) in response to adenosine diphospate (ADP) were measured and compared with platelet responses incPRP obtained simultaneously fromthe same donor.

The response of ADP stimulated platelets in VAC-PRP differs strikingly from that in cPRP. In the latter, platelets react with a dose-dependent primary aggregation, followed by a thrombin (Ila)-independent second wave of aggregation associated with 5HT-secre-tion.Platelets in VAC-PRP, however, demonstratean increased primary aggregation in responseto ADP, which is followed by a IIa-mediated second wave of aggregation and 5HT-secretion.Increasing the VAC concentration does not affect the primary aggregation response, but delayed the IIa-dependent secondary events in a dose-dependent way. At 0.5 μ M VAC, platelets react to ADP (10 μM f.c.) with reversible aggregation only. No matter this high ADP-dose, secretion reaction does not occur. At this VAC concentration, epinephrine (5 μM f.c.) does not cause aggregation and 5HT-release at all, whereas incPRP both reactions occur quite readily. Although in VAC-PRP, epinephrine retains its synergistic effect on ADP to aggregate platelets, no 5HT release was ever observed and the resulting aggregation was alwaysreversible It is concluded that VAC is a suitable anticoagulant to investigate platelet function in the presence of physiological calcium concentration. Since platelet aggregation and release appear very different from results obtained in the usual way (cPRP, low calcium concentration) the physiological meaning of this latter method needs re-evaluation. Finally, our results cast severe doubt on epinephrine as an important platelet stimulant under physiological conditions.
HEPARIN CATALYZED FACTOR XIa INHIBITION BY ANTITHROMBIN III
Authors: soons H , Jansen-claessen T , Tans G C , Hemker H C
Published: August 2018

The inactivation of human factor XIa by human antithrombin III (AT III) was studied under pseudo-first order reaction conditions (excess AT III) both in the absence and presence of heparin. The time course of inhibition was followed using SDS-PAGE. After electrophoresis proteins were blotted onto nitrocellulose and stained either for glycoprotein or for AT III using antibodies against AT III. Concomittant with factor XIa inactivation two new slower migrating bands became visible on the blots. One of these, representing the intermediate complex consisting of one AT III complexed with one of the active sites present in factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two AT III molecules. This indicates that inhibition of factor XIa by AT III can be described as:

Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity towards the chromogenic substrate S2366. Pseudo first order reaction kinetics were observed throughout. The time course of inactivation and the distribution of the reaction products observed upon gelelectrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e. independent of each other) with the same rate constant of inhibition (k1= k2)- The rate constant of inactivation for the active sites in factor XIa was found to be 1,000 M-1 s-1 in the absence of heparin and 34,900 M-1 s-1 in the presence of saturating amounts of heparin.- From the kinetic data a binding constant Kd of 0.11 uM was inferred for the binding of AT III to heparin. Experiments with four well characterized heparin fractions indicate, that the actual magnitude of the rate enhancement of factor XIa inactivation is, however, not only due to the binding of AT III to heparin, but also depends on the type of heparin to which the AT III is bound.
HEPARIN DIRECTS THE INACTIVATION OF ANTITHROMBIN BY ELASTASE
Authors: Jordan R E , Kilpatrick J , Nelson J , New gren J O , Fournel M A
Published: August 2018

In apparent contradiction to its anticoagulant activity, we have observed a previously undetected, and potentially opposing function for heparin: a distinct heparin-dependency for the in vitro inactivation of highly-purified human antithrombin by neutrophil elastase. Similar to its ability to accelerate antithrombin-mediated inhibition of coagulation enzymes, anticoagulantly-active heparin was also found to stimulate the rate of inactivation of antithrombin by the neutrophil enzyme.In the absence of heparin, or in the presence of the heparin antagonists platelet factor 4 or polybrene, little or no inactivation of antithrombin occurred. Catalytic amounts of heparin and elastase caused the complete inactivation of antithrombin (approximate molar ratio of 1:1:400 respectively) in 5-10 minutes. The loss of heparin binding affinity by the elastase-cleaved form of antithrombin permitted its separation from active antithrombin by heparin-agarose chromatography.The purified elastase-inactivated antithrombin was injected into rabbits for determination of its comparative clearance behavior. In contrast to intact, functional antithrombin (t 1/2 >30 hours) and the thrombin-antithrombin (T-AT) complex (t 1/2 previously shown to be minutes), elastase-inactivated antithrombin circulated for approximately 13 hours. This prolonged clearance relative to the T-AT complex may suggest an alternative explanation for the circulating, non-functional antithrombin observed in certain coagulopathic states. In summary, these results point to a potential and unexpected role for heparin in directing the inactivation of antithrombin and suggest a possible in vivo mechanism for neutralizing the usually non-thrombogenic nature of the vascular lining.
FIBRIN-STIMULATED RELEASE OF VON WILLEBRAND FACTOR FROM ENDOTHELIAL CELLS IS LINKED TO FIBRINOPEPTIDE B CLEAVAGE
Authors: Ribes J A , Wagner D D , Francis C W
Published: August 2018

von Willebrand factor (vWf) is synthesized in endothelial cells and stored in specialized organelles, the Weibel-Palade bodies. We have examined the role of fibrin as a potential physiological secretagogue of vWf from Weibel-Palade bodies using indirect immunofluorescence staining of endothelial cells to detect release. Addition of fibrinogen to endothelial cell cultures resulted in the formation of a clot, and this was temporally associated with vWf release. Addition to endothelial cells of preformed fibrin prepared by clotting fibrinogen with thrombin also stimulated release of vWf within 10 minutes. Hirudin inhibition or heat denaturation of clot-bound thrombin abolished most of the thrombin activity but did not diminish release. The role of fibrinopeptide A and B (FPA, FPB) cleavage in stimulating release was examined using reptilase or the venom from A. contortrix to selectively remove FPA or FPB. Release was stimulated by fibrin from which FPB had been cleaved by either thrombin or A. contortrix, while desAA fibrin prepared with Reptilase was an ineffective stimulus. The formation of a stimulatory fibrin clot with the contortrix enzyme, which does not cause release by itself, demonstrates that fibrin stimulation was completely independent of thrombin activity. The capacity to stimulate release was found to be independent of factor XIIIa crosslinking with both crosslinked and noncrosslinked FPB cleaved fibrins demonstrating stimulation. We conclude that fibrin stimulates rapid release of vWf from endothelial cells independent of thrombin activity and may function as a physiologic secretagogue. Furthermore, the stimulating capacity is dependant on cleavage of FPB suggesting that release is mediated by an active site near the N-terminal of the 3 chain or is dependent on a fibrin structure resulting from FPB cleavage.
THROMBIN INDEPENDENT TRANSGLUTAMINASE IN VASCULAR CELLS AND TISSUES MAY PROVIDE AN ALTERNATE PATHWAY TOWARD CLOT STABILIZATION
Authors: Achyuthan K E , Borowitz M J , Shuman M A , Greenberg C S
Published: August 2018

Blood coagulation Factor XIIla (FXIIIa) is a thrombin activated transglutaminase (TG) that is involved in the final step of fibrin stabilization. FXIIIa inhibits fibrinolysis by crosslinking α-2-plasmin inhibitor (α-2-PI) to fibrin. A thrombin-independent TG has been identified in vascular cells and tissues -from human, rabbit, rat, porcine and bovine sources. The vascular TG had several properties similar to the well characterized guinea pig liver TG. Both enzymes had similar molecular weights (80-90 kDa) and similar chromatographic and electrophoretic properties. Both enzymes preferentially crosslinked α-chains of fibrinogen and their TG activities were independent of thrombin treatment. Finally, both enzymes reacted with polyclonal and monoclonal antibodies to guinea pig liver TG. However, the TG from cultured adult bovine aortic endothelial (ABAE) cells exhibited a novel Ca++/Mg++ dependence for enzymatic activity which was distinct from purified liver TG. TG from confluent ABAE cells and rabbit vascular smooth muscle cells had between 4-7 fold higher TG activity compared to rapidly dividing (nonconfluent) cells -from the same passage. The difference in activity was not due to enhanced degradation of TG catalyzed isopeptide bonds by nonconfluent cells Upon examination by immunoblots using anti-TG antibodies, the TG antigen in nonconfluent cells appeared extensively degraded. Furthermore, guanosine-5'-triphosphate (GTP) was nearly 3-fold more inhibitory to TG from confluent cells compared to nonconfluent cells. Proteases, GTP and divalent cation levels may be modulating intracellular TG activity. The TG antigen detected by imm-unohistochemical techniques was predominantly associated with endothelial and smooth muscle cells of arteries, veins, venules and capillaries. TG antigen also codistributed with fibronectin antigen along the hepatic sinusoids. The ABAE cell TG crosslinked α-2-PI to fibrinogen. The modified fibrinogen was 40-fold more resistant to plasminolysis compared to unmodified fibrinogen. In conclusion, the presence of a thrombin-independent TG in blood vessels may provide an alternate pathway to inhibit fibrinolysis and promote clot stabilization.
PLATELET AGGREGABLLITY AND METABOLISM IN PATIENTS WITH UNSTABLE ANGINA PECTORIS
Authors: Terres W , Hamm C , Kupper W , Bleifeld W
Published: August 2018

Several platelet products indicating platelet activation have been detected in blood and urine of patients (PTS) with angina pectoris (AP) at rest. Platelet activation mainly depends on local changes in the morphology or biochemical behaviour of the vessels. Whether platelet hyperaggregability is of additional importance in the pathogenesis of unstable AP is up to now unclear. In a prospective trial we therefore evaluated 32 patients (PTS) with coronary heart disease, 16 with AP at rest during the last 8 hours before blood collection and 16 age and sex matched controls with stable exertional AP. Platelet aggregation was measured upon stimulation with ADP (0.5, 1 and 10 μmol/l) and collagen (1and 5μg/ml), and c-AMP was determined in platelet rich plasma before, and, as an estimate of platelet adenylate cyclase activity, after stimulation of this enzyme with PGE 1 (10 μmol/l for 30 s). For all concentrations of both ADP and collagen no significant differences in the rates and extents of aggregation could be found between the groups. Correspondingly, the mean (±. 2 SEM) concentrations of c-AMP were similar, basally (4.1 ±.1.4 pmol/ml for PTS withunstable AP and 5.3 t 1.3 pmol/ml for PTS with stable AP)and after stimulation of platelet adenylate cyclase with PGE 1 (14.8 ± 4.1 vs. 17.2 ± 2.8 pmol/ml).

Conclusion: No generalized platelet hyperaggregability could be detected in our PTS with unstable AP when compared to controls with stable exertional AP.
INCREASED PLASMA CONCENTRATION OF CROSSLINKED FIBRIN POLYMERS IN ACUTE MYOCARDIAL INFARCTION
Authors: Francis C W , Connadhan D G , Scott W L , Marder V J
Published: August 2018

Thrombin cleaves fibrinopeptides from fibrinogen, converting it to fibrin monomer, and activates factor XIII which catalyzes the formation of intermolecular e-(y-glutamyl)-lysine bonds to stabilize the fibrin polymer. The formation of factor XIIIa-catalyzed fibrin polymers during clotting of plasma and purified fibrinogen in vitro was followed using an SDS agarose gel electrophoretic technique with radiolabeled antifibrinogen antibody overlay. Prior to clot formation an increase in both total amount and sizes of crosslinked fibrin polymers was demonstrated with at least 10 distinct polymeric forms identifiable by a time corresponding to .92 of the clotting time. Soluble polymers were shown to be crosslinked through y dimer formation by two dimensional electrophoresis with proportionately more dimer in each successively larger polymer. Plasma from patients initially presenting with acute myocardial infarction (MI) showed increases in the plasma concentration of fibrin polymer and in the proportion of total fibrinogen present as polymer, as determined by a quantitative adaptation of the electrophoretic technique. The plasma concentration of crosslinked fibrin dimer in patients with subendocardial or transmural MI showed significant (p <.005) increases to 4.0 ± 1.0% and 3.6 ± .8%, respectively, as compared to the concentration in normal plasma (.8 ± .1%). No difference in plasma concentration of fibrin polymer was found in samples from patients with transmural compared to subendocardial MI. This study provides the first direct demonstration and quantitation of factor XIIIa crosslinked fibrin polymers in thrombotic disease and the findings are indicative of increased activity of both thrombin and factor XIIIa in acute MI.
COMPARISON OF ENZYMATIC PROPERTIES OF HUMAN PLASMA FVIIa AND HUMAN RECOMBINANT FVIIa
Authors: Lund-Hansen T , Peterson L C
Published: August 2018

Human plasma FVIIa (pFVIIa) and human recombinant FVIIa (rFVIIa) were both purified by immune adsorption chromatography using a calcium dependent monoclonal antibody. The FVII obtained is highly purified and contains only trace contaminants as revealed by SDS-PAGE and reverse phase HPLC chromatography. FVII was fully activated during the purification procedure. A FVIIa activity assay has been developed in microplates using human FX as a substrate and methoxycarbonyl-D-cyclohexal-alanyl-glycyl-arginine-pNA as a chromogenic substrate for the FXa generated. The assay was linear at FVIIa concentrations between 0.5 and 10 nM. The concentration of the chromogenic substrate was 0.5 mM. A pH optimum at about 8 was found. An apparent Km=0.2 μM for FX was found for both pFVIIa and rFVIIa.The results suggest that the kinetics of human FX activation by pFVIIa and rFVIIa are identical. The FVIIa activity was found to be calcium dependent with maximal activity at about 0.25 mM, while the activities at 1 and 2 mM were 20% and 3%, respectively. When rabbit brain extract is used, the well-known dramatic enhancement effect of thromboplastin could be demonstrated with both FVII preparations. Also this reaction is calcium-dependent; however, the profile of the curve is distinctly different. Poly-D-lysine (MW 160,000) was found to enhance the FVIIa activity in a concentration dependent manner. Maximum stimulation (fivefold) was obtained at a concentration of about 10 mg/l.
COMPARISON OF THE EFFECT OF FACTOR VII PREPARED FROM HUMAN PLASMA (pVIIa) AND RECOMBINANT Vila (rVIIa) IN VITRO AND IN RABBITS
Authors: Hender U , Lund-Hansen T , Winther D
Published: August 2018

Purified human FVIIa has been shown to induce haemostasis in pat. with haemoph. A compl. with antibodies against VIII:C (Hedner & Kisiel 1983). The in vitro effect of addition of pVIIa or rVIIa was studied by adding 0, 44, or 180 u pVIIa/rVIIa/ml to haemoph. A or haemoph. B plasma. The APTT shortened from 61 s (mean of 5 determin.) without any Vila added to 38 s after addition of 44 u pVIIa to haemoph. A plasma, and to 33 s after addition of 180 u/ml. In haemoph. B plasma the corresponding APTTs were 64 s, 35 s (44 u pVIIa/ml Vila) and 31 s (180 u pVIIa/ ml). Similar results were obtained when rVIIa was added to the same plasmas. In haemoph. A plasma APTT was shortened from 66 S to 52 s (50 u rVIIa/ml) and to41s(159u rVIIa/ml), and in haemoph. B plasma 71 s,44 s,and 37 s.Also in plasma from pat. with acquired antibodies against VIII:C a shortening of APTT was found both of pVIIa (60 s, 37 s, and 32 s) and rVIIs (65 s, 38 s, and 33 s). In normal plasma the APTT only shortened a few seconds after addition of the same amounts of pVIIa/rVIIa per ml (30 s, 27 s, 25 s). The doses required to normalize the APTT in vitro exceed substantially the doses used in vivo so far. The pVIIa and rVIIa were therefore also given i.v. to rabbits.Plasma samples were drawn before inj., 15 min, 2 hrs,4 hrs, 8 hrs, and 24 hrs after and platelets, APTT, fib.gen, ATIII, α2M, α2AP, ethanol gel. test, FVII,Hb, Hkr were followed. Doses of 500 u/kgb.w.,1000 or 5000 u/kg b.w. were given. No signs of a gen.activ.of the coag. were observed. The FVII level in plasm rose adequately. In conclusion higher doses of Vila than used clin. so far may be needed to achieve full haemost. effect in haemoph. pat. or pat. with acq. antibodies against VIII:C. Such doses also seem to be safe. rVIIa was as active as pVIIa and as safe in rabbits.
DRUG INTERACTIONS WITH THROMBOLYTIC AND PROFIBRINOLYTIC AGENTS: PHARMACOLOGIC STUDIES IN PRIMATES
Authors: Fareed J , Kumar A , Walenga J M , Hoppensteadt D
Published: August 2018

Thrombolytic and profibrinolytic agents have been found to exhibit varying degrees of interactions with heparin, low molecular weight heparins (LMWHs), prostacyclin and cycloxgenase inhibitors. Our initial in vitro studies did not reveal significant synergistic effects between the lytic agents and the above drugs. To study the in vivo effect of these interactions a primate (Macaca mulatta) model was employed. Initially the relative fibrinolytic and/or profibrinolytic efficacies of streptokinase (Kabi), urokinase (Choay) and t-PA (American Diagnostica) were evaluated in normal and hypercoagulable primates (homologous serum treated) in terms of both the consumption of plasminogen, c*2 antiplastin and fibrinogen, and the formation of FDP, D-Dimer and Bgl5-42 related peptides. In subsequent studies, IV and SC pretreatment of the animal with a LMWH CY 222 (Choay) at 1 mg/kg, followed by administration of t-PA (5000 u/kg IV), resulted in a marked enhancement of the fibrinolytic effects with a prolongation of the bleeding time. The results with CY 222 pretreatment followed by streptokinase (2500 IU/kg IV) or urokinase (2500 IU/kg IV) administration were similar but less dramatic. In this model, t-PA also showed synergistic interactions with urokinase and protein C concentrate. Pretreatment of primates with varying doses of urokinase and protein C prior to t-PA administration resulted in a marked decrease of anti-tPA titre and a slight increase in antigenic and functional t-PA. The augmentation in t-PA activity by these thrombolytic agents may be due to the formation of various modulatory fibrinogen/fibrin related peptides or the generation of endothelial release products. The mechanism of LMWH induced synergism of thrombolytic agents may also involve the release of endothelial t-PA or a reduction of modulatory plasma proteins. Although preliminary, these observations suggest that thromboly-tic/prothrombolytic agents exhibit varying degrees of drug interactions with themselves and with antithrombotic agents. Pre-clinical knowledge of these interactions may be of value in the design of effective polytherapy or monotherapy. Furthermore, drug interactions with thrombolytic/prothrombolytic agents should be taken into account to optimize safety/efficacy of these agents on an individual basis.
CORONARY THROMBOLYSIS IN DOGS WITH A NONGLYCOSYLATED VARIANT OF HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR LACKING THE FINGER AND GROWTH FACTOR DOMAINS
Authors: Cambier P , van de werf F , collen D
Published: August 2018

A variant of human tissue-type plasminogen activator (t-PA-AΔFE3X), with deletion of the NH2-terminal fibronectin-like finger (F) and epidermal growth factor (E) domains, and with amino acid substitution of Gin for Asn at all known N-linked glycosylation sites was expressed in Chinese Hamster Ovary Cells and purified to homogeneity. The thrombolytic and pharmacokinetic properties of this variant were studied in a canine model with copper coil induced thrombosis of the left anterior descending coronary artery. Infusion of t PAΔFE3X at a rate of 5 pg/kg/min for 30 min in 3 dogs resulted in a plateau level in plasma of 0.66 ± 0.08 ug/ml and induced recanalization of the coronary artery within 24 ± 4 min (mean ± SEM). Bolus injections over 2 min of 0.15 mg/kg in 3 dogs resulted in peak antigen levels in plasma of 1.6 ± 0.72 μg/ml and caused reperfusion within 14 ± 6 min. Bolus injection of 0.075 mg/kg in 3 dogs gave plasma antigen levels of 0.81 + 0.20 μg/ml and induced lysis in 31 ±15 min. Further reduction of the bolus to 0.038 mg/kg yielded plasma peak levels of 0.43 ± 0.20 μg/ml but did not cause reperfusion within 3 hours. Bolus injection of 0.075 mg/kg of natural t-PA isolated from melanoma cell culture fluid (Mel-t-PA) resulted in plasma peak levels of 0.46 ± 0.09 μg/ml and caused recanalization within 3 hours in only 1 of 4 dogs. None of the injections was associated with systemic fibrinolytic activation and fibrinogen degradation. The disposition of t-PAΔ related antigen from plasma following bolus injection could be described by a sum of two exponential terms with t1/2α: 17 min and t1/2β: 100 min. No significant difference in disposition rates for the different bolus injections were observed. Corresponding values for t1/2α of Mel-t-PA are 3 min.

It is concluded that the deletion mutant t-PAΔFE3X has a markedly slower disposition rate from plasma than intact t-PA, which renders it relatively more effective than natural t-PA after bolus injection.
ENDOTHELIUM CURTAILS THE TRANSFER OF PLATELET SEROTONIN INTO THE BLOOD VESSEL WALL
Authors: Bult H , Fret H , Herman A G , Jordaens F , Peeters F A M , Verbeuren T J
Published: August 2018

Blood platelets are the principal source of serotonin (5-hydroxytryptamine, 5HT) in the circulation. As it is assumed that 5HT release by activated platelets can influence vascular tone, we investigated whether platelet derived 5HT can penetrate from the lumen into the vessel wall. Venous blood was collected from pentobarbitone anaesthetized dogs on 5.8 mM EDTA, platelet rich plasma was prepared and incubated with 3H-5HT and 14c-adenine. After washing, a platelet suspension (PS, 108/ml) was made in Ca2+ free Krebs'. A segment of each saphenous vein was removed and de-endothelialized for about half (2-3 cm) of its length. The PS was perfused (37°C, 3 ml/min) for 15 min (4 dogs) or 30 min (4 dogs) through the lumen in the absence (one V. saphena) or presence (contralateral vein) of 0.1 unit thrombin/ml. Thereafter, 1 cm of the area with and the area without endothelium was excised, and these segments were washed 24 times with 5 ml Krebs. The 3H and 14c content of wash fluid and solubilized tissue was measured. The 12th wash contained 1.0 unit thrombin in order to verify that the 3H was not derived from adhering platelets. The 14c data indicated that - at low shear stress -similar platelet numbers adhere to the intima with or without endothelium, but in the absence of endothelium thrombin-induced aggregates remain loosely attached to the intima. The wash procedure removed the majority of these adhering platelets, as well as their 3H. In all tissues 3H accumulated, eg 708 dpm after 15 min and 1673 dpm after 30 min perfusion followed by washout. Both values were 7 to 10 fold enhanced when the platelet release reaction was induced with thrombin. In addition, endothelial denudation doubled the 3h accumulation under both circumstances. Previous experiments with soluble 5HT indicated that 70 % of the 3h is present in the adrenergic nerve terminals of the vessel. Application of the PS to the outside of the vessel led to a larger 3h accumulation, but then there was no indication of a difference between area's with or without endothelium.

In conclusion, removal of the endothelial metabolic barrier enhanced vascular accumulation of 5HT, but the occurrence of a release reaction appeared to be more important in determining the access of platelet derived 5HT to vascular smooth muscle cells and adrenergic nerve endings.
CONTRACTIONS TO PLATELETS IN AORTAS OF CONTROL AND CHOLESTEROL-FED RABBITS
Authors: Verbeuren T J , Van Diest M J , Herman A G
Published: August 2018

Atherosclerotic aortas obtained from cholesterol-fed rabbits show a decreased responsiveness to noradrenaline, an increased responsiveness to low concentrations of serotoninand an unaltered responsiveness to prostaglandins. In vitro contractions induced by aggregating platelets are largely due to serotonin liberated during the aggregation. The present study was designed to compare the contractile responses to aggregating platelets inaortas obtained from control and cholesterol-fed rabbits.

Male New Zealand rabbits were fed either a control or a 0.3% cholesterol diet during 16 weeks. Macroscopic and microscopic examination of the luminal surface of the aortas obtained from these animals revealed a substantial amount of fatty streaks in the tissuesobtained from the cholesterol-fed rabbits. Segments of the aortic arch of the rabbits were then mounted in organ chambers for isometric tension recording.

In both the control and the atherosclerotic aortas increasing concentrations of platelets evoked contractions; the contractions obtained with the lower concentrations of platelets were significantly greater in the atherosclerotic tissues. The maximal responses and the ED50-values were comparable in both groups of blood vessels. No significant differences were observed when platelets obtained from control or hypercholesterolemic rabbits were compared. In the control and the atherosclerotic aortas the thromboxane receptorantagonist BM13505 at 2 x 10-5M did not significantly affect the contractionsto platelets obtained from either control or cholesterol-fed rabbits. The serotonin receptor antagonist ketanserin at 5 x 10-8M nearly abolished the responses to platelets in bothgroups of aortas.

These experiments illustrate that (1) thecontractions induced by rabbit platelets in control and atherosclerotic aortas are mediated by serotonin and (2) the responses to platelets, as those to serotonin, are augmented in the atherosclerotic preparations.
RAPID AND STRONG PLATELET REACTIONS AND COAGULATION CAUSED BY INJURY OF PROLIFERATING INTIMAL SMOOTH MUSCLE CELLS
Authors: Jϕrgensen L , Grϕthe A G , Groves H M , Richardson M
Published: August 2018

We have shown that while one balloon catheter injury to rabbit aortae results in formation of a monolayer of platelets on the de-endothelialized surface, a second identical injury 7 days later gives rise to platelet-fibrin thrombi as observed at 30 minutes after the reinjury. The enhanced thrombogenicitythe second time was related to damage of theneointima which had formed during the intervening days. It was unclear whether it was due to damage of the cells or of the interstitum. In order to further explore the mechanisms of the enhanced thrombogenicity followingreinjury we observed by SEM and TEM the sequence of reactions before and during the first seconds and minutes after the second injury. The neointima was distributed around and opposite the branch orifices; the remainder of the luminal surface consisted of uncovered subendothelium. Within 10-30 seconds afterthe reinjury some of the neointimal cells were partly detached, and a few platelets and fibrin-like strands were in contact with them. Within 1-5 minutes many platelets, both single and in groups, with or without fibrin strands, had gathered in association with injured smooth muscle cells. The reinjured subendothelium remained uncovered the first 10-30 seconds; at 1-5 minutes an incomplete monolayer of swollen platelets had formed, but no fibrin was observed.These observations confirm that injury to neointima causes increased thrombogenicity. Since the first platelet reactionsand coagulation are associated with injured neointimal cells, it is likely that injury to these cells precipitates these reactions.
INCREASED FACTOR VII REACTIVITY IN THE RABBIT FOLLOWING DIET-INDUCED HYPERCHOIESTEROIAEMIA
Authors: Mitropoulos K A , Walter S J , Meade T W , Esnouf M P
Published: August 2018

The association of factor VII coagulant activity (VIIC) with plasma lipid concentrations has been a consistent feature of a number of studies in man and points to plasma lipoproteins as determinants of VIIC.To modify plasma lipoprotein concentrations and to study the effect of this on VIIC, rabbits were fed a 1%- cholesterol-supplemented diet. Treatment resulted in a many-fold increase in plasma cholesterol concentration with the major fraction of excess cholesterol associated with the very low and intermediate density lipoprotein fractions. VIIC was considerably higher in rabbits fed 1%- cholesterol-supplemented than in rabbits fed the standard diet. In both groups of rabbits, the direction and extent of variation in VIIC coincided with variation in cholesterol concentration so that over time there were significant and positive correlations between VIIC and plasma cholesterol. A method that provides a measure of the total functionalfactor VII concentration (VII) was also used. This assay involves clotting the plasma in the presence of excess tissue factor and therefore the conversion of VII tothe more reactive two-chain form of theprotein (αVIIa) .The concentration of αVIIa present in the serum was measured from the rate of activation of excess of [sialyl-3H]-bovine factor X. By day 10 of treatment, and in all furthercomparisons VTIt was only slightly higher in the group of rabbits fed cholesterol-supplemented than in that fed the standard diet.This increase in VI11 istoo small to explain the considerable increasin VIIC in the hypercholesterolaemic rabbit. We conclude thattheincrease in VIIc was to ahigher proportion of αVIIa in theplasma of hyperchol⋆esterol-aemic rabbits rather thanto an increase in the concentration of the single-chain protein.
CORNARY HEART DISEASE, DIET, SERUM LIPIDS, PLATELET FUNCTION AND PLATELET FATTY ACIDS IN TWO POPULATIONS WITH A HIGH AND A LOW INTAKE OF DIETARY FISH
Authors: Simonsen T , Vårtun Å , Lyngmo V , Nordθy A
Published: August 2018

In the coastal areas of Northern Norway the intake of fish is by tradition high whereas in the inland area it is low. We have examined the mortality of CHD in the period 1975-84 in a coastal community (C) and an inland community (I). In addition we have examined 30 healthy male subjects aged 30-year, selected by random in the two communities. The examination included a dietary survey based on registration and weighing of all dietary items for one week, blood pressure, serum lipids, primary bleeding time, platelet aggregation induced by collagen and fatty acid composition of platelet total phospholipids.

The age-adjusted mortality of CHD was significantly higher for age groups 30-70 year in C whereas the opposite was found above 70 years of age. The mean intake of fish per day was 134 g (0.9 g eicosapentaenoic acid-EPA) in C and 53 g (0.25 g EPA) in I. Serum triglycerides was higher in C (p<0.05) whereas totalcholesterol was similar. The primary bleeding time was not different in the two areas. Significantly lower concentrations of collagen was needed to induce 30 and 60% aggregation in platelet rich plasma in C than in I. No significant differences in the content of eicosapentaenoicacid (EPA) was observed in platelet total phospholipid fatty acids. This study has not confirmed that a high intake of fish as a singledietary variant, is associated with a low mortality of CHD. The lack of changes in plasma lipids, platelet fatty acid composition between representative groups from the two populationsindicate that other factors mask the possible beneficial effects of a high fish diet. Furthermore, the daily intake of large amounts of lean fish give only a very moderate increase in dietary intake of EPA.
TURNOVER OF THE PHOSPHOMONOESTER GROUPS OF POLYPHOSPHOINOSITIDES IN UNSTIMULATED HUMAN PLATELETS
Authors: Holmsen Holm , Verhoeven Adrie J M , Tysnes Ole-Bjørn , Aarbakke Grethe M , Cook Carol A
Published: August 2018

The metabolic activity of the polyphosphoinositides in unstimulated human platelets was studied by 1) short-term labelling with 32P-Pi, 2) by replacement of PP. from pre-labelled platelets with unlabelled phosphate and 3) by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphates of phosphatidylinositol-4-phosphate (PIP2) and phosphatidylinositol 4,5-bisphosphate (PIP2) had the same specific radioactivity as the γ-phosphate of metabolic ATP. The specific radioactivity of the 1-phosphates of phosphatidylinositol, PIP and PIP2 was similar but only 4-13 % as compared to the γ phosphate of ATP. When 32P-Pi. pre-labelled platelets were incubated with up to 25 mA of unlabelled phosphate, the displacement of the P-label from PIP, PIP2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in 32P-Pi pre-labelled platelets resulted in a rapid fall in metabolic ATP with much slower fall in 32P-PIP. 32P-PIP increased initially and decreased thereafter in parallel with PIP2. However, ATP turnover was not abolished, as indicated by the marked (25 % of the control) incorporation of extracellular 32P-Pi. into PIP and PIP2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PIP and PIP2 to metabolic inhibition.

We conclude that PIP and PIP2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PIP and PIP2 in unstimulated platelets is as rapid as that of the γ- Phosphate of metabolic ATP, and accounts for about 7 % of basal ATP consumption.
PLATELET-FIBRIN CLOTS FORMED BY THROMBIN SELECTIVELY RETAIN PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIF2)
Authors: Vickers John D , Kinlough-Rathbone Raelene L , Fraser Mustard J
Published: August 2018

It is established that stimulation of human platelets with thrombin for 60 s in the absence of fibrinogen increases the amount of PIPp compared with unstimulated controls (4.7 ± 0.24 nmol/109 plat, vs 3.83 ± 0.14 nmol/109 plat., p<0.01, n=8). However, stimulation with thrombin for 60 s in the presence of fibrinogen causes a large decrease in the amount of PIP2 that can be extracted with acidified chloroform/methanol compared with unstimulated controls (1.62 ± 0.39 nmol/109 plat, vs 3.84 ± 0.44 nmol/109 plat., p<0.001, n=6). Stimulation of rabbit platelets with thrombin in the presence of fibrinogen also decreases the amount of extractable PIP2 (60% at 60 s, p<0.001, n=8). Similar decreases in amount can not be demonstrated for phosphatidylinositol 4-phosphate, phosphatidylinositol, phosphatidic acid or phosphatidylcholine under the same conditions, indicating that the decrease is specific for PIP2. With rabbit platelets, polymerized fibrin formed by reptilase, which does not stimulate platelets or induce clot retraction, does not cause the decrease in extractable PIP, (3.06 ± 0.05 nmol/109 plat, were extracted compared with 3.182 ± 0.07 nmol/109 plat, without reptilase). However, stimulation of rabbit platelets with ADP in the presence of polymerizing fibrin formed by reptilase causes | larger decrease in extractable PIP2 (to 2.54 ± 0.19 nmol/109 plat., p<0.05, n=4) than is caused Dy ADP and fibrinogen alone (to 2.87 ± 0.06 nmol/10 plat., p<0.05, n=4). Inhibition by glycyl-L-prolyl-L-arginyl-L-proline of polymerization of fibrin formed by the action of thrombin prevents the large.decrease in the amount of extractable PIP2 (4.37 ± 0.30 nmol/10 plat, were extracted) from human platelets. These results indicate that the interaction of polymerizing fibrin with stimulated platelets is required for the decrease in PIP2. The decrease in extractable PIP2 seen with polymerizing fibrin can not be explained by increased degradation of PIP2 to IP3 or PIP. Thus, when human or rabbit platelets are stimulated with thrombin in the presence of fibrinogen, an association of polymerizing fibrin with the stimulated platelets occurs that leads to decreased extractability of PIP2. This may mean that PIP2 forms a specific association with pratelet proteins that are involved in clot retraction.
EVIDENCE THAT ABNORMAL PLATELET AGGREGATION IN SPONTANEOUSLY HYPERTENSIVE RATS IS LINKED WITH PHOSPHOINOSITIDES TURNOVER AND PHOSPHORYLATION OF 47,000 DALTON PROTEIN
Authors: Huzoor-Akbar H , Anwer Khursheed
Published: August 2018

We have shown earlier that abnormal platelet aggregation in spontaneously hypertensive rats (SHR) is not caused by prostaglandins (Thromb. Res. 41, 555-566, 1986). In this study platelets from SHR and normotensive (Wistar Kyoto, WKY) rats were used to examine the role of phosphoinositides (Pins) and protein phosphorylation in increased platelet activation in hypertension. Thrombin (0.05 U/ml) induced rapid hydrolysis of phosphatidylinositol-4,5-bis-phosphate (PIP2), phosphatidyl-inositol-4-phosphate (PIP), and phosphatidylinositol (PI) in (32p)-pO4 labeled platelets. However, significantly greater hydrolysis of PIP2 and PI was seen in SHR platelets than in WKY platelets (see Table). Thrombin also caused two- to three-fold increased accumulation of phosphatidic acid (PA) in SHR platelets than in WKY platelets (see Table).

Thrombin caused phosphorylation of 18,000 Dalton (P18) and 47, Dalton (P47) proteins in SHR and WKY Platelets. Significantly increased phosphorylation of P47 was seen at 5, 15, 60 and 240 seconds of incubation with thrombin in SHR platelets (60%, 68%, 98% and 91%) than in WKY platelets (13%, 37%, 44% and 47%). The extent of P18 phosphorylation was same in both SHR and WKY platelets. Aspirin (500 uM) did not affect phosphorylation of P47 or P18 in SHR or WKY Platelets. These data lead us to suggest that increased turnover of Pins and increased phosphorylation of P47 are involved in abnormal platelet aggregation in SHR (Supported in part by the COHC grant #86-01-A and the Ohio University College of Osteopathic Medicine).
PURIFICATION AND CHARACTERIZATION OF A PROTEIN C ACTIVATOR FROM THE VENOM OF AGKISTRODON CONTORTRIX CONTORTRIX
Authors: Orthner Carolyn L , Bhattacharya Prabir , Strikland Dudley K
Published: August 2018

There are two recent reports on the purification and properties of a protein C activator (PCA) from the venom of the Southern copperhead snalce. The purification of a 37,000 Mr nonenzymatic PCA (Martinoli and Stocker, Thrcmb. Res. 43, 253, 1976) as well as of a 20,000 Mr thrombin-like enzyme (Klein and Walker, Biochem. ,25, 4175, 1986) have been described. The purpose of this investigation was to purify and further characterize the PCA(s) from this vencm. A PCA has been isolated by sulphopropyl-Sephadex followed by gel filtration chromatography resulting in approximately a 100-fold purification with a 50% yield. PCA appeared as a single band on SDS-PAGE with an estimated Mr of 32,000 or 37,000 in the absence or presence of β-mercaptoethanol, respectively. High pressure gel permeation cinematography of PCA in Tris-buffered saline, pH 7.5 resulted in a single protein peak with a Mr of 39,000 which was coincident with activity. PCA was a potent activator of human protein C (PC) with a Km for PC of 0.6uM and a Vm of 0.02 sec-1. In addition, PCA catalyzed the arnidolysis of Tosyl-gly-pro-arg-p-nitroanilide (TGPRpNA) with a Km of 1.1 irM and a Vim of 66 sec-1. The rate of arnidolysis of five other pept idyl-arginyl-pNA substrates each tested at 1.0 mM was < 10% that of TGPRpNA. PCA was inhibited by nitrophenylguanidi-nobenzoate (NPGB), phenylmethylsulphonylflouride, D-phe-pro-arg-chloromethyi_ketone (PPACK) and soybean trypsin inhibitor indicating that PCA is a serine protease. The active site concentration of PCA as measured by NPGB titration was 90% that of the protein concentration. Measurement of the rate of PCA inhibition at varying levels of PPACK indicated that it had a Ki of 34uM .and an aUcylation rate constant of 0.09 min-1. PCA activation of PC was completely inhibited by CaC12 with an apparent Ki of 99uM. Since neither PCA arnidolysis of TGPRpNA nor inhibition by PPACK was affected by Ca2+, the effect of this metal was likely on the substrate PC. In summary, a PCA has been purified to homogeneity and has properties which are distinct from those reported. PCA premises to be a useful enzyme in studies of PC and its activation.
IMMUNOAFFINITY PURIFICATION OF PLASMA INHIBITOR FOR ACTIVATED PROTEIN C
Authors: Laurell M , Carlsson T , Stenflo J
Published: August 2018

Activated protein C (APC) is an important regulator of blood coagulation in vivo. In plasma this serine protease is slowly inhibited by a specific inhibitor, activated protein C inhibitor (PCI), (Suzuki et al. (1983) J.Biol.Chem. 258 , 163-168). We have now made monoclonal antibodies against PCI by immunizing with the APC-PCI complex. Positive clones were identified by solid phase immunoassay with 125I labelled partially purified inhibitor. After subcloning and expansion in mice, one of the monoclonal antibodies was immobilized on Sepharose 4B and used in the purification of the inhibitor. A two step purification procedure was deviced starting with passage of fresh human plasma over the column. Following extensive washing the inhibitor was eluted with 50 mM triethylamine- HCI,0.5 M NaCl, pH 11.0, from the column together with a small amount of high molecular weight material. After gel filtration on a column packed with AcA 44 the inhibitor appeared homogenous on SDS - PAGE. Approximatly 0.5 mg inhibitor was obtained from 200 ml of fresh plasma. The apparent Mr of the inhibitor was 57000 kDa on SDS -PAGE. The purified protein formed a complex (Mr =110000 kDa) with human APC. At the same time a band (Mr = 54000 kDa ) appeared that represented the modified inhibitor. When analyzed on agarose gel electrophoresis (75mM barbital buffer, 2mM EDTA at pH 8.7 ) the PCI migrated to the β2- region, whereas the modified inhibitor had a slightly more anodal mobility. The APC-PCI komplex migrated to the α2- region.

Two immunoradiometric assays were constructed with the monoclonal antibodies. One measured the complexes between APC and PCI while the other one measured the total amount of PCI present. These assays were used to study complex formation in buffer and plasma.