bluuebunny/crossref_metadata_2025 huggingface dataset:
CA2+-INDUCED STRUCTURAL TRANSITIONS OF THE PLATELET GP IIb-IIIa COMPLEX
Authors: Steiner B , Phillips D R
Published: August 2018

Previous studies have shown that the membrane glycoprotein (GP) IIb-IIIa complex can be reversibly dissociated by incubating platelets for 5 min at 37°C in an EDTA-containing buffer. Prolonged incubations (30 min) with EDTA, however, result in the formation of high molecular weight aggregates of GP IIb and GP IIIa. These aggregates of individual GP's neither bind fibrinogen nor support platelet aggregation, indicating that chelation of Ca2+ can affect the functional activity of GP IIb-IIIa. The present study was designed to identify conditions for the generation of functionally active GP IIb and GP IIIa. Functionally active subunits were defined as those which reformed GP IIb-IIIa complexes. The complexes were quantified by sucrose gradient sedimentation (complexed, dissociated and aggregated GP’s have different sedimentation coefficients) and thrombin hydrolysis (dissociated and aggregated GP lib are susceptible to hydrolysis by thrombin while GP lib in the GP IIb-IIIa complex is thrombin resistant). Purified GP IIb-IIIa could be dissociated by a 5 min incubation at 37°C with ≤ 10−5 M Ca2+. When the complexes were dissociated in the presence of Ca2+ concentrations below 10−6 m, the monomeric GP IIIa was converted to a slower sedimenting form; this change in structure caused it to become functionally inactive. In the presence of very low Ca2+ concentrations 10−6 M) both dissociated subunits subsequently formed high molecular weight aggregates. However, these changes in structure and loss in function could be prevented by dissociating the complexes in 10−6 M Ca2+ and immediately readding raM Ca2+ at 4°C. When this solution was warmed to 20°C, almost 70% of the dissociated subunits reformed heterodimeric complexes. Storage at 4°C for as long as 6 h did not alter the functional activity of these subunits. Octylglucoside, but not Triton X-100, completely inhibited reassociation. Experiments performed in the presence of various H+ and salt concentrations showed that the interactive forces between GP IIb and GP IIIa are both electrostatic and hydrophobic. Thus, conditions have been obtained for the preparation of functionally active GP IIb and GP IIIa which can reform the native heterodimeric complex. Various Ca2+ concentrations can have multiple effects on the structure of the dissociated subunits.
PSEUDO-THROMBOCYTOPENIA ASSOCIATED WITH AN ANTICOAGULANT INDEPENDENT IgM PLATELET AGGLUTININ IN A PATIENT WITH PROLONGED BLEEDING TIME
Authors: Horellou M H , Capelle C , Lecompte T , Kaplan C , Lecrubier C , James J M , Le Menn R , Muller J Y , Samama M
Published: August 2018

An original observation of platelet agglutination was found in a 63 y.o. man during 5 years following, without spontaneous bleeding. Low platelet counts (10 × 109/liter) and large agglutinates were observed on blood specimens collected into EDTA (1 to 15 mg/ml whole blood WB), citrate (1/10 volume citrate 0.11 M), sodium heparin (100 units/ml WB), citrate plus acetyl salicylic acid (5 to 100 mg/ml WB), citrate plus prostacyclin (Upjohn 10−5 to 10−6 M)? and buffering anticoagulant (0.8 M citrate, pH 4.65). Low platelet count and large agglutinates were also observed in capillary blood obtained by finger puncture kept at 22° or 37°C. All techniques revealed significant clumping making assessment of overall platelet number impossible. Bleeding time Ivy horizontal incision is longer than 20 minutes.

Patient's serum induced normal platelets agglutination up to a 1/512 dilution at 22°and 37°C temperature. Lack of agglutination after treatment of the serum by IgM and indirect immunofluorescence tests identified the IgM nature of the agglutinating factor. This antibody reacted with Glanzmann thrombasthenia platelets ruling out the IIb/IIIa complex as the target of this agglutinin.

There was no evidence of platelet activation following agglutination of autologous or alldgenic platelets : electronic microscopy of native blood platelets clumps did not show any sign of activation, and plasma level of B-thromboglobulin was normal in this patient. Normal platelets agglutination by patient's serum was not accompanied with ATP secretion (luciferin-luciferase).

This IgM agglutinin was associated with elevation of IgM (700 mg/dl) without clinical or biological signs of auto-immune disease.

No similar case with irreversible platelet agglutination in both capillary or venous blood has been reported before. The significance of the observation remains obscure.
CARRIER DETECTION AND PRENATAL DIAGNOSIS IN HAEMOPHILIA A BY GENE ANALYSIS
Authors: Moodie P , Peake I R , Liddell M B , Bloom A L
Published: August 2018

Restriction fragment length polymorphism (RFLP) analysis has been used to perform family studies, including prenatal diagnosis, in 21 haemophilia A kindred.Two intragenomic RFLPs were studied in conjunction with one linked RFLP. The intragenomic BgII RFLP,situation 3' to exon 26 was detected with cDNA probe C (Genetics Institute) giving bands of 20kb (17% of X chromosomes) and 5kb (83%), and the intragenomic Bell RFLP, situated 3' to exon 18, was detected with the genomic DNA probe pi 14.12 from Genentech. The frequency of this RFLP in the local population was 23% (1.1 kb allele) and 77% (0.88kb allele). The linked probe DXS15 (DX13) was used to detect a Bglll RFLPwith alleles of 5.8kb (45%) and 2.8kkb (55%). A recombination rate of approximately 5% has been estimated between the factor VIII and DXS15 loci

Carrier studies were performed in 15 kindreds. 25 obligate carriers were identified and of these, 20 were potentially informative (heterozygous and phase known) for at least 1 RFLP (9 for Bgll, 9 for Bell and 7 for BgIII). 34 possible carriers were studied, of which 13 were diagnosed as normal (6 by BgII, 6 by Bell and 5 by BgIII). 17 were diagnosed as carriers (2 by Bgll, 12 by Bell and 10 by Bglll) and diagnosis was not possible in a further 4 cases. Of these diagnosed as carriers 3 were non-informative for all RFLPs, and 14informative for at least one RFLP (3 by Bgll, 8 by Bell and 8 with BgIII).Prenatal diagnosis was attempted by analysis of DNA extracted by chorionic villussampling in 6 cases of male fetuses at risk of havinghaemophilia A. 1 fetus was diagnosed as being affected (Bell) and was electively terminated. Three otherfetuses were diagnosed as normal by the BgIII/DXS15 RFLP, but the two intragenomic RFLPs were non-informative. Because of the possibility of a crossover allthree patients opted for mid-trimester fetoscopy andmeasurement of fetal factor VIII at Kings College Hospital, London (Dr Reuben Mibashan), where the diagnoses were confirmed. In the 4th case a normal fetus was diagnosed by the Bgll RFLP analysis, but a spontaneous abortion at 12 weeks prevented confirmation of this result. In the final case of twin male fetuses, none of the RFLPs was informative and both were diagnosed as normal by fetal blood sampling at fetoscopy.
RECURRENT MUTATIONS AND AN UNUSUAL DELETION IN HEMOPHILIA A
Authors: Youssoufiän H , Patel A , Phillips D , Kazazian H H , Antonarakis S E
Published: August 2018

We have identified 15 mutations of the factor VIII (F8) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects byrestriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22, and exon 24; a single CG to TG transition was identified in exon 23; and a CG to CA transition was identified in exon 24. In addition, a Taq I site alteration in intron 4 was identified in a patient with mild hemophilia, which arose dg. S23&in a grandpaternal germ cell. Cloning and sequencing of this region suggests the generation of a newsplice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. Six different deletions were also characterized. In one family, the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.

Of the 15 mutations, 6 occurred de novo within 2 generations: 4 in males and 2 in females. In these djg.novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man.
EVIDENCE FOR A HOMOLOGY BETWEEN THE HEPARIN AND HEPARAN SULFATE BINDING REGIONS TO ANTITHROMBIN III
Authors: Fischer A M , Tapon-Bretaudière J , Dautzenberg M D , Sternberg C , Choay J
Published: August 2018

As we have previously demonstrated, heparan sulfate, a glycosaminoglycan physiologically present on the endothelial wall, is, like heparin, unable to potentiate the inhibitory effect against Factors IIa and Xa of two abnormal type 3 antithrombin III (AT III) variants. We report here that a synthetic pentasaccharide which constitutes the sequence of the heparin binding site to AT III is also unable to potentiate these two AT III variants in an anti-Factor Xa assay. According to these data, we speculated the existence of a homology between the heparin and heparan sulfate binding regions to normal AT III. We thus studied the competitive inhibition by the pentasaccharide of heparin and heparan sulfate in their potentiation of AT III activity. Such a competitive inhibition can be observed in an AT III antiFactor IIa assay because the pentasaccharide which exhibits a high anti-Factor Xa activity is devoid of any anti-Factor IIa activity. In the absence of pentasaccharide, with both heparin and heparan sulfate, a plateau is reached in AT III potentiation for concentrations of respectively 2.5 ng and 17.2 μg (corresponding to the same anti-Factor IIa activity of 0.4 u/ml for both glycosaminoglycans). In the presence of 5.5 μg of pentasaccharide, the inhibition of heparin cofactor activity is 70%. With heparan sulfate, the inhibition by the same amount of pentasaccharide is less pronounced, being only 30%. These results strongly suggest the existence of a partial homology between heparin and heparan sulfate binding sites to AT III. For heparan sulfate, the exact sequence of this site remains to be identified .
MODULATION OF ANTITHROMBIN III ACTIVITY AND ANTITHROMBIN III-THROMBIN COMPLEXES BINDING TO CULTURED CELLS BY MONOCLONAL ANTIBODIES AGAINST ANTITHROMBIN III
Authors: Knoller S , Savion N
Published: August 2018

Two monoclonal antibodies (mAb's) against antithrombin III (ATIII) were characterized with respect to their ability to interfere with ATIII activity. AT III activity was measured by its ability to inhibit the amidolitic activity of thrombin on the substrate BCP-100. Incubation of 150 ng of ATIII with 28pg mAb A36R2 prior to addition of 50 ng thrombin totally abolishes the inhibitory effect of ATIII on thrombin. Incubation of 200ng of ATIII with 10 μg of mAb B26R4 prior to addition of 75 ng thrombin raises the inhibitory effects of ATIII from 37% to 100%. We examined the effect of these mAb's on binding of antithrombin III-thrombin (ATIII-Th) complexes to bovine corneal endothelial cells. 120 pg/ml mAb's are reacted with 2 μg/ml ATIII-Th complexes prior to their addition to the cells. mAb A36R2 completely blocks ATIII-Th complexes binding. In contrast, mAb B26R4 enhances binding up to 250% of the control binding.

We conclude that mAb A36R2 prevents binding of thrombin to ATIII by recognizing an epitope on ATIII close to thrombin binding site or that its binding to ATIII induces a conformational change in the thrombin binding site thus it no longer recognizes thrombin. mAb B26R4 has a heparin-like effect on ATIII: Its binding to ATIII induces conformational changes which improve thrombin binding to ATIII. There is a correlation between inhibition and enhancement of thrombin binding to ATIII and of ATIII-Th complexes binding to cells by the two mAb's. These mAb's may provide a new tool to control the activity of ATIII and to identify the cellular binding site on the ATIII-Th complex.

This research was supported by a grant from the National Council for Research and Development, Israel and G.S.F. München, Germany.
STUDIES ON THE CONSUMPTION OF THE PLASMINOGEN-BINDING (PB) AND NON-PB FORMS OF α2-ANTIPLASMIN BY ACTIVATING PLASMINOGEN IN PLASMA WITH t-PA IN VITRO AND IN VIVO
Authors: Leebeek F W G , Kluft C , Rijken D C , Knot E A R , McNeill A , Cohen A F
Published: August 2018

α2-Antiplasmin (AP) occurs in blood in two functional forms: a plasminogen binding (PB) and a non-PB (NPB) form with differences in the mechanism of plasmin inhibition. The two forms can be assayed using modified crossed immuno-electrophoresis with plasminogen incorporated in the first dimension gel. We studied the consumption of both AP-forms by activating plasminogen with t-PA: In vitro by addition of one-chain t-PA to plasma and in vivo in patients receiving two-chain rt-PA (BW-t-PA) during myocardial infarction. Results demonstrate that at various dosages of t-PA (up to 3 μg/ml plasma or 100 mg/90 min) after 90 min specifically the PB form was consumed (up to 95%), while the NPB form remains essentially unchanged (decrease ≤ 15%). Also during longer incubation the NPB form was not decreased. The consumption of the PB form was accompanied by the formation of plasmin-AP (PAP) complexes, which could be precipitated by antibodies to AP. SDS-PAGE followed by immunoblotting showed the complex and the post-complex peptide of about 14 kD immunochemic-ally related to AP. Also agarose-electrophoresis resulted in the separation of the peptide from the PAP complex. It showed a high electrophoretic mobility, which could be strongly reduced by the presence of plasminogen and it is therefore identified as the C-terminal part of AP, harbouring the plasminogen binding site of AP. Other experiments indicate that in plasma the C-peptide remains bound to the PAP complex: Upon gelfiltration the peptide co-chromatographs with the PAP complex and in vivo the peptide remains in circulation as long as the PAP complex is present in plasma. It is concluded that upon activation of plasminogen in vitro in plasma and in vivo during thrombolytic therapy with t-PA preferentially the PB-form of AP becomes consumed with the formation of a PAP complex. The PAP complex harbours a non covalently attached C-terminal peptide. Apparently the NPB form becomes not complexed to plasmin even during extensive systemic effects of t-PA in vivo.
MECHANISM OF PLASMINOGEN ACTIVATION AT LOW TEMPERATURE IN PLASMA SAMPLES CONTAINING THERAPEUTIC CONCENTRATIONS OF t-PA
Authors: Rijken D C , Seifried E , Barrett-Bergshoeff M M , Kluft C
Published: August 2018

It is known that plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may continue during plasma handling, leading to artificially low fibrinogen (Fbg) and α2-antiplasmin (AP) values. Addition of D-Phe-Pro-Arg-CH2Cl or quenching antibodies against t-PA prevents this phenomenon, but these additions do not allow measurement of t-PA activity. The question of this study is, why the in vitro effects occur, even during freezing of the samples. Normal plasma was supplemented with various amounts of two-chain melanoma or recombinant t-PA and stored at -20°C, with and without a prior snap-freeze procedure. AP consumption (chromo-genic substrate assay) and Fbg degradation (Clauss method), measured in thawed samples, were most pronounced in the non snap-frozen samples. As it took a relatively long time before these samples were really frozen, the time course of the effects was studied at different temperatures. Plasma samples containing 1000 IU t-PA per ml were incubated at 37, 25,10, 0 and -8°C between 0 and 120 min. AP reduction was most rapid at 37°C (50% after 13 min), was less at 25°C (50% after 30 min), but did not further decrease at lower temperatures. The AP reduction at temperatures between 25 and -8°C corresponded to the effect of 40% t-PA activity at 37°C. The Fbg values gave a similar picture: the most rapid reduction occurred at 37°C, a slower reduction at 25°C, but no further reduction (even a small increase) was found from 25 to -8°C. The experiments were repeated in a purified system, consisting of t-PA, plasminogen, Fbg and AP. In contrast to the plasma system, AP reduction gradually decreased from 37 to 0°C. The apparent t-PA activity at 0°C was 6% of the activity at 37 °C.

It is concluded that the in vitro effects in plasma samples containing t-PA can be, at least partially, explained by an abnormally strong plasminogen activation around 0°C. A normal temperature dependency in the purified system strongly suggests that unknown plasma factors enhance plasminogen activation at low temperatures.
THE BINDING OF TISSUE PLASMINOGEN ACTIVATOR TO PLATELETS
Authors: Loscalzo J , Vaughan D E
Published: August 2018

Since the platelet surface has been shown to be a site for plasminogen conversion by tissue-type and other plasminogen activators, we examined the binding of tissue plasminogen activator (tPA) to human platelets. Resting, washed platelets were found to bind single chain, radioiodinated, recombinant tPA specifically and saturably with an apparent, estimated dissociation constant (KD) of 458 nM, binding approximately 570 molecules per platelet at saturation. Washed platelets activated with adenosine 5'-diphosphate in the presence of 0.1 mg/ml fibrinogen and 1 mM CaCl2 bound tPA with greater affinity, having an estimated apparent KD of 30.6 nM and binding approximately 29,000 molecules per platelet at saturation. Bound tPA could be completely displaced by an excess of unlabeled tPA. Interestingly, bound tPA could also be displaced from activated platelets with increasing concentrations of soluble fibrin with an estimated IC50 of 37.5 μg/ml of fibrin. In contrast, increasing concentrations of fibrinogen failed to reduce binding. These data show that tPA binds to the activated platelet surface by a mechanism that involves platelet-bound fibrinogen. In addition, these data suggest that on binding to the platelet surface, fibrinogen expresses domains that are similar to the tPA binding domains of fibrin. It is the presence of these domains within the platelet aggregate that likely supports tPA binding and facilitates plasminogen activation.
CELLULAR CATABOLISM OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HIGH AFFINITY UPTAKE SYSTEM ON RAT HEPATOCYTES
Authors: Bakhit C , Lewis D , Billings R , Malfroy B
Published: August 2018

The uptake, internalization and intracellular degradation of 125I-labeled rt-PA (125I-rt-PA) by isolated rat hepatocytes was investigated. Incubation at 37°C resulted in internalization of 125I-rt-PA, followed by the appearance of labeled trichloroacetic acid-soluble (TCA) material in the inclubation media due to degradation of rt-PA. Degradation of rt-PA was inhibited by the presence of NH4Cl (10mM) or chloroquine (ImM) (lysosoma tropic agents) in the incubation media. This suggests that rt-PA degradation occurs intracellularly, perhaps within the lysosomes. 125I-rt-PA was taken up by rat hepatocytes through a specific, high affinity mechanism. Scatchard analysis of the data indicated that 106 molecules of rt-PA were taken up per cell/hour and the calculated dissociation constant was lOnM. Uptake of 125I-rt-PA was not inhibited by glycopeptides isolated from rt-PA nor by several other glycoproteins known to be cleared by identified hepatic receptors. These results suggest that the uptake of rt-PA by rat hepatocytes involves a receptor specific for t-PA and is not mediated by a carbohydrate specific receptor.
FIBRINOLYTIC ACTIVITY OF ARMS AND LE6S IN PERIPHERAL VENOUS HYPERTENSION IN MAN
Authors: Carriero M R , Annoni F , Mussoni L , Cerletti C , de Gaetano G
Published: August 2018

Spontaneous fibrinolytic activity of venous specimens Is greater In the arms than in the legs of normal subjects. This difference might be caused by the different hydrostatic pressure In arms and legs. We tested, on standard fibrin plates, the fibrinolytic activity of euglobulins prepared from venous blood obtained from arms and legs of normal subjects and patients with chronic peripheral hypertension. Normal subjects (26-38 yrs old, n=5) were tested both before and after 10 min venous occlusion (V0) of an arm and after 10 min occlusion of a leg. V0 was obtained by applying the cuff of a sphlgmomanometer at a pressure value Intermediate between systolic and diastolic pressure. Patients (39-64 yrs old, n=7) were tested both before and after V0 of the arm and after 10 min orthostatic posture (mean 100 mmHg). For each Individual the fibrinolytic activity In the arm before V0 was considered as basal value of both the arm and the leg. In normal subjects fibrinolytic activity Induced by V0 was greater In the arm than In the leg (262.9°74.9 versus 165.5°52.9 mm2). The average Increase of fibrinolytic activity after V0 was 3.4 (arms) and 2.1 (legs). In patients with peripheral venous hypertension fibrinolytic activity was 298.3°46.7 mm2 In the arm and 131.1 °19.2 mm2 In the leg. The average Increase Induced by VO In the arm was 3.5 while the activity of the legs after orthostatic pressure was 1.6. In conclusion patients with peripheral venous hypertension did not show any reduced fibrinolytic response after VO of the arms. Fibrinolytic activity in patients" legs after orthostatic pressure was also similar to that In the legs of volunteers after venous occlusion.
EVIDENCE FOR THE OCCURRENCE IN HUMAN PLASMA OF A COMPLEX OF UROKINASE (UK) AND PLASMINOGEN ACTIVATOR INHIBITOR TYPE I (PAI-1)
Authors: Dooijewaard G , Gillis C P M , van der Hoeven R J , Rijken D C , van Hinsbergh V W M
Published: August 2018

In plasma three forms of UK may occur: proUK, active UK and an inactive complex of UK with inhibitor(s). Recently we developed 1. an ELISA which measures the antigen of the three forms with equal efficiency and gives the total UK, irrespective of its molecular form, and 2. a BIA, which measures the activity of the immuno-immobilized material before (UK) and after activation with plasmin (proUK + UK) . With these assays the concentrations of proUK, UK and UK.Inhibitor complex in plasma were estimated to be 2.1, < 0.1 and 1.1 ng/ml, respectively. The identity of the inhibitor involved remained unexplained. In this study we investigated the possibility that it is PAI-1. First we titrated PAI-l-containing serum-free culture medium of endothelial cells with increasing amounts of UK, measured the concentration of the remaining free UK by BIA and calculated the concentration of UK.PAI-1 formed. A linear and proportional relationship between UK.PAI-1 and the amount of medium titrated was established. Then we analysed the obtained titration curves in the ELISA and found closely overlapping straight lines with no evidence for any antigen to have been titrated away. This confirms that the UK.PAI-1 formed, indeed, is measured in the ELISA with equal efficiency as the free UK and affirms the possible presence of UK.PAI-1 in plasma. Hereafter we set out for the development of a UK.PAI-1 ELISA, which first singles out the UK antigen from the plasma, then senses the PAI-l-containing part with anti-PAI-1 antibodies conjugated with biotin and thereafter detects the concentration with streptavidin-HRP. Calibration curves were made with in vitro prepared UK.PAI-1 added to UK immuno-depleted plasma. Preliminary data give 0.8 ng/ml for the UK.PAI-1 in human plasma, which explains the bulk of the 1.1 ng/ml UK.Inhibitor complex.
PLATELET AGGREGATION DYNAMICS TO ADENOSINE DIPHOSPHATE IN NON-STIRRED SUSPENSIONS: LONG-RANGEINTERACTIONS FOR HUMAN, BUT NOT RABBIT, PLATELETS
Authors: Longmire K , Frojmovic M M
Published: August 2018

The simplest experimental approach for a theoretical description of platelet aggregation is based on kinetics of early multiplet formation (‹4 platelets per aggregate)occurring with diffusion-dependent particle collisions (no flow). The Smoluchowski theory was used to calculate collision efficiencies, αβ, from a linear plot of platelet particle count (Nt)−1 vs time (t) following addition of adenosine diphosphate (ADP) to citrated platelet-rich-plasma (PRP) for 7 human (H) and 2 rabbit (R) donors. A 0.1 ml sample of PRP was stirred with ADP for 0.5s, then immediately transferred to a 37°C bath for no-stir (diffusion) studies or further stirred with ADP for stir-induced aggregation studies. Samples were fixed with 0.5 ml 0.8% glutaraldehyde with particle count (Nt) determined with a resistive counter and % aggregation (PA) computed (reproducibility/sensitivity ‹ 5%). For stir conditions, R platelets were as sensitive and as rapidly aggregated by ADP (2-10 μM) as H platelets, with ∼ 1 s time lag for onset of PA. However, for no-stir conditions, linear regression analysis of data for ADP (5-10 μM) induced PA for H platelets for 0-30 s gave αβ = 7.5±4.6 (r = 0.9±0.05). Analysis at longer “diffusion” times showed a second phase (60-300 s) in some H donors with aB = 0.5±0.4 (4/9 donors), while R platelets showed only 1 phase with αβ = 0.65±0.15 (0-60 to 0-900 s) (r = 0.8±0.1). The ADP sensitivity ([ADP]½ corresponding to 50% of maximal changes) for the abnormally rapid PA in no stir H PRP for early times, measured over 0.4-100 μM range, was found to be ∼9 μM (5-17 μM range) and 3.5 μM (3-10 μM) for measurements respectively at 5-10 and 20-30s; these values were ∼ 3-8 × greater than lADPji measured for stirred suspensions for rate/extent of PA or rate of turbidometrically-measured macroaggregation (TA), while › [ADP] threshold for secondary aggregation in TA (10 H donors). These abnormally large aB values and their ADP sensitivity observed for human platelets are consistent with long-range interactions mediated by“chemotactic” agents released from the cells but distinct from normal dense granule release requiring macroaggregation, or by as yet uncharacterized membrane or polymetric bridges.
REQUIREMENT FOR THROMBIN RECEPTOR OCCUPANY DURING PLATELET SECRETION UNDER AGGREGATING CONDITIONS
Authors: Koike Kazuo , Holmsen Holm
Published: August 2018

We have previously showed that hirudin abruptly arrests thrombin-induced secretion of acid hydrolase at any stage of its progress, whereas it only affects dense granule secretion only at its initial stages; these results have been interpreted to show that acid hydrolase secretion requires sustained while dense granule secreion ony requires a brief receptor occupancy (Holmsen et al. JBC 256(1981)9393). The requirement for receptor occupancy in thrombin-induced α-granule secretion and secretion during aggregation have been studied. Increasing concentrations of thrombin were added to gel-fitered platelets containing a constant, high concentration of hirudin. Dense granule secretion was initiated at lower thrombin concentration than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 14-fold exess of hirudin produced abrupt stop of dense granule secretion and α-granule secretion when added to platelets shortly after thrombin; it had no affect after these secretory process had reached 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, all three secretory processes increased their rates and could now be abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule andoαgranule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum. It is concluded thatαgranule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for hydrolase secretion.α-granule secretion might, however, require longer occupancy than dense granule secretion. It is possible that aggregation potentiates all secretory responses through close cell contact and that the abrupt inhibition by hirudin of all secretions may have been caused by its effect on the slower aggregation.
Thrombin stimulation of human platelets:Phosphoinositides as the only source of the diacylglycerol moiety in phosphatidic acid
Authors: Tysnes O-B , Verhoeven AJ M , Holmsen H
Published: August 2018

Recent reports on various cell types including platelets have concluded in the heterogenous production of phosphatidic acid (PA) during the stimulus-response coupling. Human platelets were pulse-labelled simultaneously with [32P] P. and [32H]glycerol. Extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. When the cells were stimulated with low concentrations of thrombin, the production of [32P] PA was evident without measurable production of [3H] PA. At higher doses of the agonist,[3H] PA was formed, but distinctly later than [32P] PA.

This suggested a heterogenous production of PA upon thrombin stimulation of platelets. The specific H-radioactivity of PA in unstimulated cells was about 50% of that of the phosphoinositides. Upon l80 sec of stimulation with 0.5 U/ml of thrombin, the specific [3H] PA radioactivity increased to the level of the phosphoinositides which remained constant during platelet activation. Since other phospholipids incorporate [3H] glycerol much slower than the phosphoinositides, these latter remain the only possible source of the diacylglycerol moiety of PA. The specific 32P-radioactivity of PA in unstimulated cells was only 4% of that of α-ATP and similar to the specific 32P-radioactivity of phosphatidylinositol in unstimulated platelets. After Jyijin of stimulation with 0.5 U/ml of thrombin, specific [32P] PA was similar to that of γ-ATP. The descrepancy in [32P] Pi. and [3H] glycerol incorporation into PA upon thrombin stimulation of platelets is therefore mainly due to a thrombin-induced shift inspecific 32P-radioactivity in PA.
WHOLE BLOOD AGGREGOMETER IN THE ASSESSMENT OF PLATELET HYPER-AGGREGABILITY
Authors: Abbate R , Boddi M , Favilla S , Costanzo G , Paniccia R , Paniccia G F
Published: August 2018

The aim of this study has been to investigate the reliability of platelet aggregation in whole blood in some clinical conditions associated to thromboembolic complications.

18 healthy subjects, 15 patients affected by ischemic heart disease (IHD) and 15 patients affected by insulin independent diabetes, free of vascular complications, were studied. Collagen induced (2.5 mg/L f.c.) platelet aggregation was evaluated both in whole blood (WB) by using impedance whole blood aggregometer (Chrono-Log) and in platelet rich plasma (PRP) by Born aggregometer. Aggregation was significantly higher in whole blood than in PRP in all the groups investigated (p < 0.01). No significant difference was found in PRP aggregation among the three groups, whereas WB aggregation was significantly higher in the two patient groups (IHD 79.5 + 14.2%, Diabetes 81.3 + 17.6%) than in controls (64.8 ± 14.1%) (p < 0.01 for both comparisons). No relationship was found between WB aggregation and Hct or platelet number in any of the groups studied. A slight relationship was found between megathrombocyte count and WE aggregation values (r=0.31, p < 0.05).

Collagen platelet aggregation in WB seems to be provided with higher sensibility than PRP aggregation in detecting hyper-aggregability, probably because it does not imply the selection of platelet populations with loss of larger platelets and of other blood cells.
MEASUREMENT OF PLATELET IONIZED CALCIUM IN PATIENTS WITH MYELOPROLIFERATIVE DISORDERS BY AEQUORIN METHOD
Authors: Fuzimoto T , Fuzimura K , Kuramoto A
Published: August 2018

In myeloproliferative disorders(MPD), bleeding tendency and thrombosis are encountered occasionally in the presence of high platelet counts. It has been reported that there are some abnormalities of membrane glycoprotein or arachidonate metabolism in platelets of MPD. We measured the intracellular change of calcium levels[Cai2+] after stimulation in platelets of the patients with MPD by Aequorin method. Eleven cases of chronic myelogenous leukemia(CML), 7 cases of polycythemia vera(PV), 4 cases of essential thrombocythemiaCET^ and 12 normal adults were studied, and as stimulators 0.5 U/ml thrombin and 2.5 μg/ml collagen were used. In the patients with CML, PV and ET, maximum intracellular calcium level was significantly lower and the reaction period reaching to the peak calcium level(the arrival time) was significantly prolonged than the normals after thrombin stimulation. On collagen stimulation, maximum[Cai2+] level in patient with CML and PV was significantly lower and the arrival time in patient with CML and ET was significantly prolonged than the normals. No correlation was found between maximum [Cai2+] level and maximum aggregation rate or platelet counts in those patients. The increasing rate of intracellular calcium levels after stimulation with A23187 in the presence of various concentration of extracellular calcium was proved lower in CML than in the normal. [45Ca2+] uptake rate into the CML platelets after thrombin stimulation showed lower rate compaired with normals. These results suggest that platelets in patients with MPD have some abnormalities of calcium influx mechanism. We already reported that platelet membrane glycoprotein (GP)IIIa was increased significantly in MPD platelets. The study is going to examine the relationship between this membrane abnormality and impaired calcium influx in MPD.
IN VIVO POTENCY OF HEPARIN AND HEPARINOIDS TO INHIBIT RAT SMOOTH MUSCLE CELL PROLIFERATION
Authors: Dryjski Maciej , Mikat Eileen , Bjornsson Thorir D
Published: August 2018

The final response of endothelial cell injury in the arterial wall is characterized by proliferation of smooth muscle cells (SMC) in the intima to form a fibro-musculo-elastic plaque. Recent in vivo and in vitro studies have shown that heparin can inhibit proliferation of SMC. These studies, however, have not elucidated the relationships between heparin dose or concentration and its in vivo antiproliferative response. In the present study, we investigated the potency of standard heparin (SH), low molecular weight heparin (LMWH) and a mixture of sulfated glycosaminoglycans (Organon 10172) with respect to the in vivo inhibition of SMC proliferation after endothelial cell injury. The injury was achieved by a short infusion of air into an isolated segment of the rat carotid artery. The drugs were administered by the AlzetR miniosmotic pumps for two weeks, at which time the animals were sacrificed and both carotid arteries were fixed in situ for light and transmission electron microscopy. The index of the intimal SMC proliferation was the maximum intima to media area (I/M) ratio. The control animals developed a marked intimal thickening (I/M: 0.97). The animals treated with 50 units/kg/hr of SH exhibited significantly less intimal hyperplasia (I/M: 0.10). With decreasing SH doses, there were increases in the I/M ratio (5 units/kg/hr, I/M: 0.44; 0.5 units/kg/hr, I/M: 0.75, and 0.05 units/kg/hr, I/M: 0.84). LMWH in doses of 50 units/kg/hr inhibited SMC proliferation as effectively as SH (I/M: 0.10), however, at doses of 15 units/kg/hr the I/M ratio was 0.55. The effect of Organon 10172 was significant at doses of 50 units/kg/hr (I/M: 0.04), but limited at doses of 15 units/kg/hr (I/M: 0.61). The APTT and anti-Xa levels were only slightly increased in the animals treated with 50 units/kg/hr of LMWH and Organon 10172, but unchanged in the animals receiving SH and the lower doses of LMWH and Organon 10172. It is concluded that SH, LMWH and Organon 10172 have significant antiproliferative effects upon SMC. The differences in the dose-response curves suggests more than one mechanism of action.
SITES IN TISSUE-TYPE PLASMINOGEN ACTIVATOR INVOLVED IN THE INTERACTION WITH FIBRIN, PLASMINOGEN AND LOW MOLECULAR WEIGHT LIGANDS
Authors: Verheijen J M , Caspers M P M , de Munk G A W , Enger-Valk B E , Chang G T G , Pouwels P H
Published: August 2018

Tissue-type plasminogen activator (t-PA) activates the proenzyme plasminogen to the active protease plasmin which degrades fibrin. The unique properties of t-PA, fibrin binding and stimulation of activity by fibrin make it an interesting molecule for specific thrombolysis. t-PA is thought to consist of five structural regions designated finger (F), growth factor (G), kringle 1 (Kl), kringle 2 (K2) and protease (P). Previous studies have shown that the interaction of t-PA with fibrin is mediated by the F and K2 regions.

Mutated t-PA cDNA molecules were expressed in Chinese hamster ovary cells and t-PA analog proteins were purified from serum free culture media using affinity chromatography with immobilized monoclonal antibodies. Besides FGK1K2P (native t-PA) the following analogs were used GK1K2P, klK2P, K2P, P, FP and FGKlk2P (kl and k2 have partial deletions of the kringle). All the molecules comprising K2P could be stimulated in plasminogen activation activity by fibrinogen fragments comparable to normal t-PA. The activities of FP and FGKlk2P were only slightly influenced by these fragments. It was shown that the fibrin binding site in K2 was plasminogen dependent whereas that in F was not. K2 was found to contain a binding site for lysine, 6-amino-hexanoic acid but also 6-amino-hexane and thus to differ from the high affinity lysine binding sites in plasminogen.

Chemical modification of lysine and arginine residues in t-PA with citraconic anhydride and cyclohexanedione respectively, revealed no involvement of these residues in interaction with lysine or analogs nor in stimulation of activity by fibrinogen fragments. Arginine modification led to inhibition of plasminogen activation activity, both in the presence and absence of fibrinogen fragments, but the amidolytic activity as measured with a tripeptide paranitroanilide was not changed. The involvement of one or more arginine residues in interaction of t-PA with plasminogen seems likely.
CHANGES IN PROTEOGLYCAN AND SULFATED PROTEIN SYNTHESIS DURING MEGAKARYOCYTE MATURATION IN VIVO
Authors: Schick B P , Walsh C J , Jenkins-West T
Published: August 2018

We investigated changes in sulfated proteoglycan (PG) and sulfated protein synthesis during megakaryocyte (MK) maturation in vivo by characterizing the (35S)-labeled molecules in MKs and platelets (PLTs) obtained daily from 3 hr to 5 days after injection of guinea pigs with (35S)sulfate. Radioactivity in macromolecules was maximal in MKs 3 hr and in PLTs 3 days after the injection. The cells were solubilized in 8M urea/50mM Tris/0.2% Triton X-100/0.1M NaCl, and PGs and sulfoproteins were separated by DEAE-Sephacel chromatography. PGs (65% of cell 35s) were eluted as two fractions, one (PG-1, 87%) with 4M Gdn HC1 and another (PG-2, 13%) with 4M Gdn HCl/2% TX-100. The Kav of PLT PG-1 on Sepharose CL-6B shifted gradually from 0.18 to 0.10 from 1-5 days after (35S) injection, and the smaller and larger PG-1 species were resolved on SDS-PAGE by fluorography. The size of PG-1 molecules was a function of glycosaminoglycan (GAG) chain length. The appearance of the different size PG-1 molecules in PLTs was accounted for by their disappearance from MKs over the same time period. Thus the size of the PG-1 synthesized by MKs decreased with MK maturation. The (35S)-PG-2 appeared in PLTs only 2-3 days after (35S) injection, had Kav 0.07 on CL-6B, but had GAGs of the same average size as those of PG-1. The hydrophobic character of PG-2 suggests that it might be the membrane PG. PG-1 and PG-2 were separated by SDS-PAGE and identified by fluorography. The core proteins of PG-1 and PG-2 were obtained by chondroitinase digestion and identified by SDS-PAGE and fluorography. The GAGs of PG-1 and PG-2 were almost entirely chondroitin-6-sulfate. The average size of PG-1 was 200,000 and its GAGs about 45,000.

The sulfated proteins (20-25% of total cell 35S) eluted in the wash-through of the DEAE-Sephacel column and with 0.23M NaCl. Their isoelectric points were 4.0-6.5. They eluted as a small peak near the V0 and a major broad peak from Kav 0.3-0.6 on CL-6B columns, and could be identified as at least 8 distinct bands on SDS-PAGE by fluorography. Digestion with NaOH/NaBH4, Pronase or papain released small (35S)-labeled fragments, and the (35S) appeared to be associated with oligosaccharides. The sulfoproteins appeared in PLTs primarily 2-4 days after (35S) injection, and different proteins were labeled at different time points.
IONOPHORE-INDUCED METABOLISM OF PHOSPHOLIPIDS AND ARACHIDONIC ACID (AA) IN PORCINE AORTIC ENDOTHELIAL CELLS (PAEC): RELEASE OF AA FROM ALKENYL-LINKED PHOSPHATIDYLETHANOLAMINE (PE)
Authors: Deykin D , Jakubowski J A , Brown M L
Published: August 2018

We have recently reported that endothelial cells incorporate and store substantial amounts of AA in ether phospholipids. Our studies demonstrated that after initial incorporation, AA is transferred from diacyl phosphatidylcholine (PC) to both diacyl and alkenylacyl PE. (Wey HE, Jakubowski JA, Deykin D: BBA 878:380, 1986) The purpose of this study was to examine the relative roles of ether lipids and diacyl lipids as sources of AA released from PAEC phospholipids following A23187 stimulation. Confluent cultures of PAEC were prelabeled with luM [14C]AA for 24 hours. The cells were stimulated with luM A23187 for time intervals between 30s and 30min. Cellular lipids were separated into major lipid classes and phospholipid subclasses (diacyl; akenylacyl; alkylacyl). The external medium was analyzed for released labeled eicosanoids. The time course of total release of 14C-radioactivity was biphasic: (1.5%/min for the first 5min; 0.6%/min 5min - 30min). The changes in cellular phospholipids were also dichotomous. From 30s - 5min there were substantial losses of [14C]AA from diacyl PE and phosphatidylinositol, but an abrupt increase in net diacyl PC radioactivity occurred. These changes coincided with the appearance of 14C-labeled prostanoids in the medium. Later changes (5 - 30min) included a sustained, progressive loss of 14C-radioactivity from alkenyl PE and diacyl PC. These changes coincided with the appearance of 14C-labeled lipoxygenase-derived products in the medium. Our data demonstrate that alkenyl PE, which contains large amounts of AA, is a potential source for biologically active eicosanoids. Although unequivocal assignments cannot be made, the data suggest that specific pools of AA provide precursors for individual classes of eicosanoids.
FAMILIAL PROTEIN S DEFICIENCY WITH A VARIANT PROTEIN S MOLECULE IN PLASMA AND PLATELETS
Authors: Schwarz H P , Heeb M J , Lottenberg R , Roberts H , Griffin J H
Published: August 2018

Deficiency of protein S (PS), the cofactor for the antithrombotic protease, activated protein C (APC), was first described in 1984 in families with venous thrombotic disease. We describe here a PS deficient family with venous thrombotic disease presenting an abnormal PS molecule in plasma and platelets. The propositus, 20 years old, and two older siblings suffered from severe venous thrombosis and pulmonary emboli documented by imaging techniques. All laboratory studies were normal except for PS. The propositus while taking oral anticoagulant had a PS antigen (ag) level of 17% and PS functional activity of > 5% as measured by the ability of APC to prolong the clotting time of a modified APTT assay using PS-immunodepleted plasma. One brother had PS ag of 42% and PS activity of 7%. As demonstrated by immunoblotting using SDS gels, both the propositus and this brother presented an abnormal PS molecule in plasma at 65,000 apparent MW versus normal PS at 70,000 apparent MW. The mother had normal PS levels (93% ag/100% activity) but had both normal (70,000 MW) and variant (65,000 MW) forms of the PS molecule in plasma as well as in platelet lysates. One clinically affected PS heterozygous deficient brother (64% ag/11% activity), two asymptomatic siblings (68% ag/9% activity and 104% ag/114% activity) and the asymptomatic PS heterozygous deficient father (59% ag/10% activity) had only normal PS molecules (70,000 MW) on PS immunoblots. Two dimensional immunoelectrophoresis studies showed that the variant PS bound to C4b-binding protein in plasma. Since PS is in platelets and megakaryocytes and synthesized by the latter cells, immunoblotting analysis of platelet lysates was done and showed that platelets of each family member contained the same pattern of normal and variant PS forms as found in plasma. This is consistent with the hypothesis that PS gene expression is similar in those cells, presumably megakaryocytes, hepatocytes and endothelial cells, that control the synthesis of both platelet and plasma forms of PS.
FACTOR XIII (FXIIIa) OF BLOOD COAGULATION NORMALIZES COLLAGEN SYNTHESIS OF SCLERODERMA FIBROBLASTS
Authors: Paye M , Krieg T , Lapière Ch M
Published: August 2018

Progressive systemic scleroderma (PSS) fibroblasts display, in some cases, an excessive collagen production which leads to fibrosis of the skin and internal organs. Administration of FXIIIa has been reported to be beneficial to some of the PSS patients. The effect of FXIIIa on collagen synthesis by PSS fibroblasts was studied in vitro in two different culture conditions: in a confluent monolayer on plastic and in a three-dimensional collagen lattice.

Proteins and collagen synthesis was measurfed by metabolic labeling for 24 h with 3H-proline in absence or presence of FXIIIa (1 U/ml) in dermal fibroblasts from an active lesion (PSS forearm), from an uninvolved area of the skin of the same patient (control abdomen) and from the skin of a normal subject.

Proteins synthesis was similar for the three strains under both culture conditions while collagen synthesis was strongly increased in PSS forearm fibroblasts as compared to the uninvolved and to the normal skin fibroblasts. The addition of FXIIIa repressed collagen synthesis of PSS forearm synthesis to the level observed in the abdominal skin fibroblasts of the patient and the normal cells in absence of FXIIIa. When cultured in a collagen lattice collagen synthesis was repressed in a similar proportion in all fibroblasts. Addition of FXIIIa further reduced collagen biosynthesis in the active PSS fibroblasts. The addition of FXIIIa in the lattice largely increased the degradation of newly synthesized collagen in all the strains of fibroblasts.

The action of FXIIIa on collagen biosynthesis was also tested in monolayer in 3 other strains of PSS fibroblasts and 3 controls. In all fibroblasts, collagen biosynthesis was reduced by 75 % after addition of FXIIIa.

Our results suggest that the beneficial effect of FXIIIa administration in PSS patient might be related to a reduction o1 excessive collagen production and perhaps to an increased degradation of newly synthesized molecules.