bluuebunny/crossref_metadata_2025 huggingface dataset:
THE EFFECTS OF THE PAI DERIVED FROM HUMAN PLATELETS ON TWO DIFFERENT TYPES OF t-PA
Authors: Yasukouchi T , Fujie T , Sakurama S , Satoh M , Ikeo M , Nakagawa S
Published: August 2018

We studied on the effect of PAI derived from human platelets on two different kinds of t-PAs; one was purchased from BioPool, Sweden, (human uterus or melanoma cell derived one-chain t-PA: one-chain u-mt-PA) and another was kindly supplied from Sumitomo Pharm. Co., Osaka, (recombinant two-chain t-PA: two-chain rt-PA). As the PAI-rich solution derived from platelets. We used the platelets extract, which was prepared as follows: The concentrated platelet-rich plasma (Red Cross Japan) was washed with phosphate buffer containing 0.4 % Triton X-100. The inhibitory activities of the PAI were measured by the method of parabolic rate assay and were calcurated from the activities remained in the mixtures of PAI and the t-PAs. The reactions of the PAI and the t-PAs were also analysed by the method of fibrin autography. The PAI suppressed the activity of two-chain rt-PA completely within 5 minutes, but could not suppressed that of one-chain u-mt-PA completely within such a short incubation time. These facts also recognized on fibrin autography. The molecular weights of both free t-PAs were recognized at 60 kDa and that of the complex of the PAI and two-chain rt-PA was recognized at 110 kDa, respectively. The fibrinolytic zone of the complex of PAI and one-chain u-mt-PA, however, could not be recognized. This indicates two possibilities; one is too small amounts of complex forming to detect and another is that SDS can not reveal the t-PA activity from the complex of PAI and one-chain u-mt-PA on fibrin autography. It remained unclear that the difference of the reactions of these t-PAs to PAI had been induced from whether the difference between one- and two-chain, or that between rt-PA and u-mt-PA. If the former is reasonable, it can be said that the molecular form of t-PA has an important significance in physiological fibrinolytic mechanism.
A TUMOR SERINE PROTEASE INHIBITOR WHICH MAY FUNCTION AS A TISSUE PLASMINOGEN ACTIVATOR INHIBITOR
Authors: Cheung A , Hebert K F
Published: August 2018

A protease inhibitor has been isolated from human colorectal adenocarcinomas by extraction with a low ionic strength-buffer and a combination of concanavalin A-Sepharose, Sephadex G-200, DEAE cellulose and chromatofocusing steps. The preparation appeared to be homogeneous upon gel exclusion chromatography and SDS polyacrylamide gel electrophoresis. It had an apparent Mr of ~66,000 and its isoelectric point was 4.6-4.7. The inhibitor was able to bind and inhibit urokinase, plasmin, trypsin, tissue plasminogen activator and thrombin. The bindings appeared to be stoichiometric and relative fast and diisopropylfluorophoshphate interfered with the bindings. However tPA was the only enzyme that was inhibited in a linear concentration-dependent manner.The inhibitor crossreacted with antiserum against al-antitrypsin but not with antisera against β2-macroglobulin, α2-antiplasmin, antithrombin III, or Cl-inhibitor. Whereas αl-antitrypsin completely inhibited the amidolytic activity of elastase, the tumor inhibitor had no effect on elastase under the same conditions.
QUANTIFICATION OF ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) ANTIGEN BY AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Authors: MacGregor I R , Booth N A , Hunter N R , Bennet B
Published: August 2018

An ELISA to measure PAI-1 antigen has been developed using PAI-1 purified from human endothelial cell conditioned medium and a monospecific antiserum raised against it in the rabbit. Test and standard samples diluted in assay buffer containing non-immune rabbit serum were incubated in microplate wells coated with anti-PAI-1 IgG. Then biotinylated anti-PAI-1 IgG was added to the wells followed by a streptavidin-biotinylated horseradish peroxidase complex. Tetramethylbenzidine was used as substrate and the optimised ELISA had a detection limit of 0.2 ng PAI-1 ml-1 sample, using purified PAI-1 of known concentration as a standard for the assay.

PAI-1 antigen was readily detectable in human plasmas and higher concentrations were invariably detected in the corresponding whole blood sera.α2antiplasmin and antithrombin III which have been shown to have high degrees of sequence homology with PAI-J were undetectable in the ELISA at concentrations of 10 ug ml-1 . Sera from baboon and Rhesus and Cynomologus monkeys exhibited partial cross reactivity in the ELISA while no crossreactivities were observed with a wide range of non-primate sera that included dog, goat, cow, horse, pig and rat.

A variety of human cell cultures were assayed for PAI-1 antigen. Endothelial cells from umbilical cord veins and arteries and from adult sapgenous veins secreted, typically,1-2 ug PAI-1 24-1 hr per 10 cells. This represented approximately 2-4% of the total secreted protein. Detectable but considerably lower levels of PAI-1 were secreted by keratinocytes and fibroblasts as well as a lung carcinoma cell line A549.No PAI-1 was detected in media conditioned by two melanoma cell lines, a breast carcinoma cell line MCF7 or a monocytic leukaemia JIII. The ELISA proved suitable for monitoring altered cellular PAI-1 metabolism, and in conjunction with functional assays, for investigating changes in PAI-1 specific activity.
A NOVEL MICROTITER PLATE ASSAY FOR FUNCTIONAL PLASMINOGEN-ACTIVA-TOR INHIBITOR (PAI) LEVELS IN WHOLE PLASMA
Authors: Carroll R C , HAnsard R M , GoldMan M H
Published: August 2018

Currently, the most widely used functional assay for PAI levels in plasma is that developed by Chmielewska et al (Thromb. Res. 31:427-436, 1983). A major drawback of this assay is the considerable manipulation of plasma samples required by the necessity of acidification and reneutralization in order to determine remaining activator levels based on plasminogen to plasmin conversion. We have devised an assay based on microtiter plates coated with 20 U/ml urokinase. In this assay, aliquots of citrate- or EDTA-anticoagulated plasma are incubated in the coated wells for 30 min at room temperature to allow PAI in the plasma to complex with the solid state urokinase. The plasma is then removed, the wells extensively washed with Tris-saline buffer plus 1 mg/ml bovine serum albumin, and the remaining urokinase activity determined by adding plasminogen and S-2251 chroma-genic substrate for plasmin. Inhibitor levels can be conveniently expressed as percent inhibition relative to the urokinase activity in wells not exposed to plasma. Absolute units of inhibitor have also been derived by titration of plasma samples with known units of urokinase (calibrated by S-2444 chromagenic substrate for plasminogen activators). Between the range of 90% to 10% inhibition, a linear slope of 0.69±0.6 units ml-1/10% inhibition is obtained.In agreement with previous reports, we find a wide variation in control subjects and some tendency towards higher PAI levels in older age groups. Age group 20-39, n=29, 36.6 ± 14.9%. Age group 40 and over, n=16, 48.9 ±12.9%.Reproducible values (SEM ± 4.6%,n=5)are obtained for control* individuals sampled and assayed at various times over a period of several months. We have also assayed plasma samples from patients undergoing a variety of surgical procedures, and have followed rapid post-surgical increases in PAI levels with a gradual return to pre-operative levels. Evidence is presented that the post-surgical increase is predominantly from the platelet releasable pool.
PARALLEL VARIATIONS OF PLASMA INSULIN, TRIGLYCERIDE AND PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI 1) LEVELS IN OBESE NON DIABETIC SUBJECTS
Authors: Juhan-Vague I , Vague P , Alessi M C , Atlan C , Valadier J , Badier C
Published: August 2018

We had shown in non diabetic healthy subjects with Body Mass Index (BMI) varying largely, a significant correlation (r) between PAI activity levels and BMI : (r=0.66) and insulinemia (r=0.52).

PAI levels were then studied in non diabetic Obese women (0)(n=50) and in age matched healthy women with normal BMI (N) (m ± SEM) = 0 versus N : BMI : 33.4± 0.8/ 20.2± 0.8 ; plasma insulin (Ins -uu/ml) : 22.7±1.5/12±1; Triglyceride(TG-mmol/1):1.240.09/0.8±0.1.The low value of euglobulin fibrinolytic activity (EFA) in Obese was due to high levels of PAI 1 : 0 versus N : EFA (mm) : 5.2 ±0.3/ 9 ±0.3 ; PAI activity (PAIact.- Verheijen's method-u/ml) :14 ± 2.1/5.04 ±0.6 -(p 0.01). In 10 0 and 10 N PAIact and PAI 1 antigen (PAI 1 Ag- Kruithof's method-ng/ml) were determined in parallel : 0 versus N : PAIact.: 29.4±3.8/ 5.8±0.9 ; PAI 1 Ag : 100±14.2/19.7 ±1.7, (p0.01). In Obesegroup correlations between, PAIact. and BMI (r=0.51), and insulin (r=0.69) and Triglyceride (r=0.48) were significant (p 0.01).

When in Obese subjects, insulinemia was decreased by 24 hours Fast (n=10) or by 15 days Metformin treatment (1.7g/day) (n=9), PAI activity (measured on fibrin plates)decreased significantly in a parallel way : compared to initial values -after 24 hours Fast =Ins : 75 %, PAIact. 73% -after Metformin treatment=Ins :75%, PAIact. :57%, TG :73%.

From these results, a direct action of insulin on the synthesis cells of PAI 1 could be evoked. An effect of Triglyceride levels cannot be excluded, the variation of Triglyceride, insulin, PAI levels being parallel.As hyperinsulinemia, hypertriglyceridemia are risk factors for atherothrombosis, as there is a link between insulin, triglyceride, PA Inhibitor 1 levels, the pathogenic role of hyperinsulinemia and hypertriglyceridemia in the development of atherothrombosis could be in part mediated by plasma hypofibrinolysis due to high level of PAI 1.
A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY
Authors: Booth N A , Reith A , Bennett B
Published: August 2018

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.

Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.
PLASMINOGEN ACTIVATOR INHIBITORS IN NORMAL PREGNANCY AND PATIENTS WITH PREECLAMPSIA
Authors: Estellés A , Gilabert J , Aznar J , España F , Fernández J A
Published: August 2018

Plasminogen activator inhibitor (PAI) activity and other fibrinolytic parameters have been studied in three groups of preeclamptic patients: A) severe preeclampsia, B) mild preeclampsia and C) chronic hypertension with superimposed severe preeclanpsia. These parameters were compared to those observed in a group of normal pregnant women at similar gestation age ( 3rd trimester of pregnancy, group D). The PAI activity, regardless of whether single-chain t-PA or two-chain t-PA was used, was significantly higher in cases of severe preeclanpsia than in cases of mild preeclanpsia and normal pregnancy

This increase in PAI activity normalizes in early puerperium. When PAI activity was evaluated in the platelet lysate no increase in severe preeclanpsia, as compared to the control group, was observed. Following the addition of purified two chain t-PA to plasma of severe preeclanptic patients, a complex with a molecular weight of about 115,000 was detected by a zymographic fibrin technique. When urokinase (90% of HMW and 10% of LMW) was used, two complexes of about 95,000 and 85,000 were detected. The increased level of PAI activity found in severe preeclanpsia was partially inhibited by the antisera against PAI-1 and PAI-2.
PLASMINOGEN ACTIVATOR INHIBITOR OF PLACENTAL TYPE IN PREECLAMPSIA
Authors: de Boer K , Lecander I , ten Cate J W , Borm J J J , Treffers P E
Published: August 2018

In 24 patients with preeclampsia and in 24 normal pregnant controls, matched for gestational age, fibrinolytic parameters were determined. Aim of the study was to investigate plasminogen activator inhibitory activity (PAI activity) levels and plasminogen activator inhibitor of placental type (PAI 2) in preeclamptic and normal pregnancy and to establish the clinical relevance of these assays. PAI activity was measured by titrating the plasma with single chain t-PA. PAI 2 was measured by means of an ELISA using monoclonal and polyclonal antibodies against the placental inhibitor. Therefore we related testresubts to a predefined relevant clinical endpoint i.e. fetal outcome. The following results were obtained:

PAI 2 levels below 43% had a positive predictive value of 91% for poor fetal outcome, levels above 43% had a negative predictive value of 95%. PAI activity levels didn't have a significant predictive value.. It is concluded that high levels of PAI activity in preeclampsia are not due to PAI 2 and that low levels of PAI 2 are related with a poor fetal outcome.
ADP-BINDING SITES IN PLATELETS: CHARACTERIZATION BY PHOTOAFFINITY LABELING AND BINDING STUDIES WITH FIXED PLATELETS
Authors: Jefferson J R , Harmon J T , Jamieson G A
Published: August 2018

Attempts to photoaffinity label platelet ADP receptors with 2-azidoADP have not been successful possibly due to the absence of a spacer arm between the nucleotide and the photolabile group. We have synthesized a probe having a long spacer arm by coupling 2-(3-aminopropylthio)-ADP to succinimidyl 4-3H-azidobenzoate. Labeling competable by ADP could not demonstrated with intact platelets. With isolated platelet membranes, three bands (Mr 140,000, 110,000 and 46,000) were labeled that were not competed by ADP while three other bands (Mr 188,000, 92,000 and 51,000) were competable by 100 uM ADP.

Another problem in characterizing ADP receptors has been complications due to ADP metabolism and secretion from the dense granules. To avoid this problem we have measured the binding of ADP and analogues to formalin-fixed platelets. ADP bound to two sites (Kl, 0.35 ± 0.04 uM; R1, 160,000 ± 20,000 sites/platelet; K2 7.9 ± 2.0 uM; R2, 400,000 ± 40,000 sites/platelet) with low non-specific binding: these values are in agreement with ADP concentrations required for activation. Affinity at the high affinity site was in the sequence ADP(0.35 uM)=ATP(0.4 uM)›2-MeS.ADP(6.8 uM)› GDP(49 uM) › AMP(360 uM); adenosine did not compete. Binding at the high affinity site was blocked by pMBS (EC50 250 uM) and 5-fluoro-sulfonylbenzoyladenosine (EC50 1 mM). This is the first report of photoaffinity labeling of putative ADP receptors. Our experiments with fixed platelets suggest that they may be useful in testing agonists, antagonists and inhibitors in the absence of complications due to secretion and metabolism.
ADP-INDUCED CYTOPLASMIC CALCIUM MOBILIZATION AND SHAPE CHANGE IN PLATELETS ARE MEDIATED BY DIFFERENT BINDING SITES
Authors: Koneti Rao A , Kowalska Maria A
Published: August 2018

Platelet stimulation with ADP results in a number of responses including increase in cytoplasmic ionized calcium concentration [Ca2+]i, shape change, aggregation, secretion, and inhibition of cAMP accumulation caused by PGI2.

5'-Fluorosulphonylbenzoyladenosine (FSBA), which covalently labels ADP binding site on platelets, blocks platelet shape change but not inhibition of cyclic AMP levels by ADP, while p-chloromercuribenzenesulfonate (pCMBS), a non-penetrating thiol reagent, blocks ADP-induced inhibition of adenylate cyclase but not shape change. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i to determine whether it is linked to the binding site mediating shape change or that for inhibition of adenylate cyclase. In platelets loaded with Ca2+ indicators, quin 2 or fura 2, and in presence of adenosine deaminase (AD), FSBA (50-200 μM) induced a dose-dependent, rapid rise in [Ca2+]i. from basal levels of 70-90 nM to peak levels of 300-500 nM in the presence of 1 mM external Ca2+ providing direct evidence that FSBA is a platelet agonist. The [Ca2+ ]i. returned to near basal levels over 30 min. The effect of FSBA on [Ca2+]i. was inhibited by ZK 36,374 (40 nM), a stable PGI2 analog. AdP concentrations eliciting similar responses were about 10-fold less than those for FSBA. Platelet incubation with FSBA (50-100 μM) in the presence of AD for 30 min (to ensure optimal covalent labelling of the ADP binding sites) abolished shape change but jjid not inhibit ADP (5, 25 μM)-induced increase in [Ca2+]i. or block the inhibitory effect of ADP on cAMP accumulation in1platelets exposed to ZK 36,374 (50 nM) in.presence of theophylline (7 mM). Incubation with pCMBS (5-100 pM, 2 min) abolished the effect of ADP on [Ca2+]. and on the inhibition of cAMP levels; shape change was not 1 inhabited even at 1 mM. pCMBS (0.5-1 mM) inhibited the rise in [Ca2+ ]. by FSBA alone. These observations suggest that ADP-induced Ca mobilization is mediated by platelet binding sites which are distinct from those mediating shape change but probably the same as those modulating adenylate cyclase.
TWO SIZES OF THROMBIN RECEPTORS IN SOLUBILIZED HUMAN PLATELET MEMBRANES
Authors: Harmon J T , Jamieson G A
Published: August 2018

Thrombin receptors of two functional sizes (106 and 30,000) have been previously identified by the technique of radiation inactivation (Harmon and Jamieson, Biochemistry 24:58, 1985). To determine whether different s-ized populations are separable biochemically, platelet membranes solubilized in Triton X-100 were applied to a Sepharose 6B column after incubation with 125I-thrombin. Apart from unbound 125I-thrombin (40,000) the elution profile showed two radioactive peaks with apparent mol. wts. of 1−2×106 and 170,000. The bound radioactivity associated with these two peaks could be competed by nonradioactive thrombin. Chromatography was then carried out in the absence of added thrombin and the specific thrombin binding activity of the eluted fractions was determined by a soluble radioreceptor assay (Harmon and Jamieson, J. Biol. Chem. 261:13224, 1986): two peaks with apparent mol. wts. of 1−2×106 and 170,000 were again observed. Western blotting with polyclonal rabbit anti-glycocalicin antibody showed that intact glycoprotein lb (GPIb) was associated with the high mol. wt. peak while a fragment of GPIb (Mr 120,000) was associated with the lower mol. wt. peak. Thus, in solubilized platelet membranes, thrombin binding activity exists in two mol. wt. forms (1−2×106 and 170,000) and both are associated with GPIb-like molecules. Other membrane components associated with these complexes remain to be determined.
COMPARISON OF THE PLATELET MEMBRANE SIGNAL TRANSMISSION INDUCED BY THROMBIN OR THE mAb ALB6 (CD9)
Authors: Rendu F , Boucheix C , Lebret M , Soria C , Levy-Toledano S
Published: August 2018

A CD9 monoclonal antibody described to aggregate human platelets was studied on different platelet functions in order to determine its mechani^i of action. The mAb ALB6 induces a transient decrease in P-phosphatidylinositol-4,5 bisphos-phate (PIP2) after a lag phase of 35 sec. This results in a synthesis of P-phosphatidate (PA), calcium mobilization as measured by quin 2 and phosphorylation of myosin light chain (P20) and of 43 kDa protein (P43). All these biochemical steps allow the release of dense granule, alpha-granule and lysosome constituents. Final biological and metabolic effects of thrombin and ALB6 thus appear similar but three main differences bring additional information.(1) The kinetic of thrombin-induced effects is faster. (2) The thrombin-induced release of dense granules compared to alpha-granules and lyso-somes occurs extremely rapidly (50% of the maximum C 5-HT releasable at 5 sec) and is considerably slowed down by the presence of EDTA (50% release of 14C 5-HT at 20 sec.), whereas the release of the three types of granules occurs concomitantly in the case of ALB6 -induced aggregation whether EDTA is present or not. (3) Myosin light chain is phosphorylated in both cases but external calcium is necessary for maximum phosphorylation by thrombin since it is inhibited by EDTA, which is not the case with ALB6.

The ALB6-induced lag phase would result from the fact that the signal transmission has to occur from the external non integral membrane CD9 antigen molecule and the internal phospholipase C. The different requirements in external calcium for the responses induced by the two types of inducers suggest that the early myosin light chain phosphorylation and dense granule release induced by thrombin coulcL be at least partly dependent on the entry of external Ca2+ through a putative Ca2+ channel.
THROMBIN-INDUCED BIPHASIC Ca2+TRANSIENT DETECTED BY FURA-2 FLUORESCENCE WAS COUPLED WITH BIPHASIC Ca2+ UPTAKE IN PLATELETS
Authors: Koyama M , Katabami F , Matsuno K , Matsumiya H , Abe K , Sakurada K , Ozasa S , Maekawa I , Miyazaki T
Published: August 2018

In the last congress on Thrombosis and Haemostasis in SanDiego we presented the biphasic Ca2+transient in thrombin-stimulated platelets detected by quin2. This time we tried to measure Ca2+ transient in platelets using other Ca2+indicators such as fura-2 and aequorin. In order to evaluate the contribution of increased Ca2+ influx across the plasma-membrane in elevating cytosolic free Ca concentration on platelet activation, 45
Ca2+uptake was measured simultaneouslyby silicon-oil centrifugation method with or without 5 mM EGTA treatment. Such EGTA treatment was intended to remove the entire 45Ca
2+bound to the external surface, and the values obtained accounted for the true amount of 45
Ca2+ uptaked by platelets. RESULTS: (1) When the platelets preincubated in Ca2+ poor medium were exposed to 0.1-1.0 mM external Ca2+ a variety of response of Ca2+ transient were observed in this resting state; a sharp and transient peak appeared in aequorin luminescence, while biphasic increase with an initial burst for 6 to 8 s was demonstrated in fura-2 fluorescence. 45
Ca2+uptake measurement resulted in a biphasic response, the time-course of which was correlated well with that of fura-2 fluorescence. (2) Thrombin-induced Ca2+transient was also observed. A remarkable biphasic increase of fluorescence was recognized in fura-2 assay, which showed a sharp and large response during initial 10 s after activation followed by a wide peak for the next 40 s. (3) Although total Ca2+ uptake in thrombin-stimulated platelets without EGTA treatment resulted in the simple convex-shaped increase in platelet 45Ca
2+content, that observed after EGTA treatment showed a distinct biphasic pattern; an initial small uptake during the first 10 s after activation followed by another uptake for the next 40 s. CONCLUSION: (1) Fura-2 measurement is the most excellent method avairable for monitoring intracellular Ca2+transient since the response of 45Ca
2+uptake treated by 5 mM EGTA reflect that of cytosolic Ca2+ concentration detected by fura-2 both in resting and in thrombin-stimulated platelets. (2) Membrane-lipid metabolism such as polyphosphatidilinositol breakdown or arachi-donate cascade may be involved in the mechanism concerning each phase of these Ca2+ mobilization.
PHOSPHORYLATION OF THE OUTER SURFACE OF PLATELETS ENHANCES THE EFFECTS OF COLLAGEN ON PLATELET AGGREGATION, ATP RELEASE, CALCIUM TRANSLOCATION AND PHOSPHOINOSITIDE HYDROLYSIS
Authors: Chiang T M , Wojcikiewicz R J H , Kang A H , Fain J N
Published: August 2018

We have recently isolated and purified from human plasma two isomeric forms of protein kinase, both of which can phosphorylate the outer surface proteins of human platelets. One of the proteins phosphorylated is the platelet collagen receptor. The phosphorylation of the outer surface proteins of human platelets increased their functional responsiveness to collagen. Collagen-stimulated platelet aggregation, release of ATP and calcium translocation were all enhanced by pretreatment with plasma protein kinase in the presence of ATP. The mechanism by which phosphorylated platelets become hypersensitive to collagen is not established. In the present study, we have used [3H]myo-inositol-labeled human platelets to investigate the possible role of phosphoinositide metabolism in mediating this hypersensitivity. Formation of inositol mono-, bis-, and trisphosphate in response to collagen was more pronounced in phosphorylated platelets than controls. these results indicate that enhanced phosphoinositide hydrolysis in phosphorylated platelets correlate with the increased functional responses to collagen.
COLLAGEN-DERIVED OCTAPEPTIDE (OP) AND INITIAL EVENTS OF PLATELET ACTIVATION
Authors: Karniguian A , Rendu F , Grelac F , Legrand Y J
Published: August 2018

A collagen-derived OP was shown to specifically inhibit collagen induced platelet aggregation, fibrinogen binding and prothrombinase activity, suggesting that it affects on the iniating mechanism. In view of this, early biochemical steps were investigating in platelet response to collagen and OP. In the absence of external calcium, a transient decrease in 32Pphosphatidy14,5,bisphosphate (PIP2) occuring in parallel with the shape change, but partially preceding it, was followed by a rapid resynthesis up to 25%. A slightly delayed hydrolysis of PIP also occured within seconds of platelet aggregation. Phosphatidate (PA) was produced only at a small extent , even with high doses of collagen. The phosphorylation of myosin light chain (P20) was minimal, whereas, the phosphorylation of the 43K protein (P43) occured and increased inparallel with the onset of secretion and aggregation, and was collagen dosedependent. Pretreatement by OP reduced the platelet responsiveness and parallely the P43 phosphorylation up to 80%.At concentrations allowing shape change but not aggregation, the breakdown of PIP2 persisted, and only the resynthesis was affected; high doses of OP which prevented any shape change, no PIP2 cleavage was observed.PA formation and P20 phosphorylation were slightly inhibited by 30-50% only at these high doses which completely abolished the PIP2 breakdown.

Thus OP could inhibit P43 phosphorylation and platelet aggregation at a concentration which did not affect the ' PIP2-specific phospholipase C. This suggest that collagen can activate proteine kinase C, independently of the initial PIP2 breakdown.
INTERACTION OF PLATELETS WITH DIFFERENT ISOTYPES OF HUMAN COLLAGEN: THE COLLAGEN-RECEPTOR AND SIGNAL-RES-P0NSE COUPLING:
Authors: Misselwitz F , Norden C , Domogatsky S P , Repin V S , Heine H
Published: August 2018

Platelet-collagen interaction is important in primary hemostasis and thrombosis. However, little is known about the interaction of platelets with genetically distinct collagen types I, III, IV, and V (Cl, ClII, CIV, CV) in their triple helical form. Here we report on the interaction of gel- filtered platelets with surfaces coated with monomeric collagen isotypes and the binding of 125-iodine-labeled Cl to platelets. It was shown that the binding of monomeric Cl is dose and time-dependent, saturable and specific, since it is competitively inhibited by unlabeled Cl, but not by unlabeled CV. Scatchard analysis reveals a receptor-class with a Kd of 2.5 x 10−8M. The result suggests that the different collagen isotypes may bind to platelets through various specific binding sites. Confirming that, we demonstrated, that interaction of platelets with collagen-coated surfaces is associated with the activation of different intra- and extracellular signal transduction systems: Initial attachment of discoid platelets to a CV-substrate does not induce neither synthesis of prostanoids nor changes of cyclic AMP-level. Adhesion and massive spreading of platelets on a CIV-substrate result in a decrease of stimulated cAMP-level without any synthesis of prostanoids. Only thrombus-formation during interaction of platelets with Cl- or CHI-coated surfaces is accompanied by a parallel decrease of cAMP and massive synthesis of platelet prostanoids. Additionally, it was shown that the stable prostacyclin analogue, carbacyc-lin, inhibited thrombus-formation and platelet spreading, whereas the PGH2/TXA2-receptor antagonists, 13-Azaprostanoic acid ana BM 13.177, completely inhibited thrombus-formation without any effect on platelet spreading.
EVIDENCE FOR TWO DIFFERENT Ca2+TRANSPORT SYSTEMS ASSOCIATED WITH PLASMA AND INTRACELLULAR HUMAN PLATELET MEMBRANES
Authors: Enouf J , Breadux R , Bourdeau N , Levy-Toledano S
Published: August 2018

The regulation of Ca2+ concentration in different cells involves two Ca2+ pumps. The presence of such mechanisms in human platelets is still controverted. We then investigated this question by using plasma and intracellular membranes obtained after simultaneous isolation by centrifugation ca 40% sucrose from a mixed 100,000 g membrane fraction.

The Ca2+ uptake by the different membrane vesicles has been studied. Both membrane fractions took up Ca2+ and the Ca2+ transport systems exhibited a high affinity towards Ca 2+.

However, the two associated Ca2+ transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate, while the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes.

Two Ca2+ activated ATPase activities are associated with the isolated membrane fractions and appeared different for the following parameters : 1) a different time course of the two enzyme activities; 2)a similar apparent affinity towards Ca2+ (10−7 M), though inhibition of the Ca2+ ATPase activity was only observed in intracellular membranes at 10−6 M Ca2+ ; 3)a different pH dependence with a maximum at pH 7 for the enzyme of intracellular membranes and pH 8 for the enzyme of plasma membranes; 4)a 10 fold difference in the ATP requirement of the enzymes, thus the maximal response was obtained with 20 uM for the intracellular membrane enzyme and with 200 uM for the plasma membrane enzyme ; 5) a different affinity for various nucleotides as energy donors with a higher specificity of the plasma membrane enzyme towards ATP ; 6) a different vanadate inhibition-dose reponse which did not exceed 60% for the plasma enzyme while it reached 100% for the intracellular enzyme.

Taken together, these studies agree with the possible role of both a plasma membrane and a dense tubular system Ca2+ -ATPases in the regulation of Ca2+ concentration in human platelets.
MONOCLONAL ANTIBODIES RECOGNISING PROTEINS OF THE OUTER AND INNER SURFACE OF THE PLATELET PLASMA MEMBRANE
Authors: Wilkinson J M , Hack N , Thorsen L I , Thomas J A
Published: August 2018

Platelet membrane preparations can be fractionated into two major subpopulations by free flow electrophoresis and these have been shown to correspond to the plasma membrane and the endoplasmic reticulum of the platelet. The plasma membrane fraction can be shown, by two-dimensional electrophoresis, to contain the major surface glycoproteins together with considerable amounts of actin and actin-associated proteins such as the 250 kDa actin-binding protein (filamin), P235 (talin), myosin, α-actinin and tropomyosin (Hack, N. … Crawford, N., Biochem. J. 222, 235 (1984). These cytoskeletal proteins are associated with the cytoplasmic face of the plasma membrane and probably interact with transmembrane glycoproteins. We have raised monoclonal antibodies to the purified plasma membrane preparation in order to investigate the nature of these glycoprotein-cytoskeletal interactions. In two fusion experiments, out of 804 tested, 104 hybrids secreted antibody to the membrane preparation and of these 24 were selected for further study. Initial assays were by ELISA using either the membrane preparation or whole fixed platelets as the target antigen. The specificity of the antibodies was investigated further by immunoblotting of SDS gels of total platelet proteins prepared under reducing and nonreducing conditions, by immunofluorescence, by immunohisto-chemistry and by crossed immunoelectrophoresis. The majority of the antibodies recognise major surface glycoproteins; of these, four bind to glycoprotein Ib under all conditions examined while another seven recognise the glycoprotein IIb/IIIa complex as detected by crossed immunoelectrophoresis. Three antibodies recognise the actin binding protein and these cross-react with the smooth muscle protein filamin in a number of different species. Further characterisation of these antibodies in both structural and functional terms will be presented.

We are grateful to the Smith and Nephew Foundation for financial support for these studies
PLATELET INDUCED VASOSPASM
Authors: Drouet L , Pignaud G , Baladier L , Paris C
Published: August 2018

A new model of experimental thrombosis on 100 to 300 micron diameter rat mesenteric arteries has been designed.

The focal specific deendothelialisation is induced by a tunable dye pulsed laser. All changes in the vascular lumen and of the vascular wall are observed under a stereozoom magnifier. This model allows in-time observation and quantification of induced phenomena after digitalisation and image processing of the video signal of the observation system. Besides thrombus formation and possibilities of its pharmacological manipulation, we focussed this study on the downstream vasospasm induced by platelet activation during thromboformation. The vasospasm is platelet dependent because it can be observed only when a platelet thrombus takes place . The vascular reaction is mediated through the endothelium, as the endothelium has been localy injured only on a 50 micron diameter spot at the place where the thrombus developpes and is intact at the level where the spasm is observed (more than 500 micro downstream). The role of platelet factors putatively involved in the vascular reaction has been evaluated by the use of specific inhibitors or antagonists (namely of ADP, serotonin, catecholamins, thromboxane A2 and PAF acether). Proofs are given that at least both platelet serotonin and thromboxane A2 are actively involved. These two intermediaries would sequentially participate : serotonin in the early phase and thromboxane A2 in the secondary phase. Vascular synthesis of prostacyclin does not seem to be a major intervenant in the regulation of the vascular tone.
PRESENCE OF VWF IN VASCULAR ENDOTHELIUM BUT NOT PLATELETS FROM CONTROL DOGS AND VIIIR:AG-DEFICIENT DOGS
Authors: Meyers K M , Wardrop K J , Helmick C M , White F P
Published: August 2018

Blood vessels from control and VIIIR:AG-deficient dogs (plasma VIIIR:AG was <5% of control) were surgically removed, cryosectioned, airdried and processed for immunohistochemical detection of VWF using a monoclonal antibody against human VWF, a polyclonal antibody against human VWF, and a polyclonal antibody against canine VWF. All antibodies were monospecific for canine VWF. The secondary antibody was conjugated to fluorescein or rhodamine. A biotin-avidin system was also used. Each antibody detected VIIIR:AG in veins, venules, and arterioles from control and VIIIR:AG-deficient dogs. Immunofluorescence was observed only in areas of the vessel occupied by endothelial cells. There was no detectable difference in the distribution of VIIR:AG or the fluorescence intensity in vessels from control and VIIIR:AG-deficient dogs. Platelets were isolated by gel filtration, adhered to glass slides, fixed, and then incubated with antibodies to VWF. As positive controls, human platelets were processed simultaneously. VWF could not be identified by immunohistochemistry in platelets from control and VIIIR:AG-deficient dogs. Human and canine platelets were also washed and lysed by 5 freeze/thaw cycles or by using the French Pressure Cell Press. The lysate from canine platelets did not have VWF detectable by an ELISA.
EXOGENOUSLY ADDED DIACYLGLYCEROL (DAG) AND ELEVATED INTRACELLULAR Ca2+ LEVELS CANNOT SUBSTITUTE FOR PROSTAGLANDIN END0PER0XIDES/ THROMBOXANE ki IN MEDIATING WEAK AGONIST-INDUCED PLATELET SECRETION
Authors: Krishnamurthi S , Kakkar V V
Published: August 2018

We have compared the abilities of exogenously added U46619, the PG endoperoxide analogue and, sn-l-oleoyl 2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (diCg), the membrane-permeant DAG analogues, at restoring weak agonist-induced secretion in indomethacin (10μM)-treated platelets (I-PL) in the absence of endogenous PG/Tx synthesis. [14C]-5HT secretion from pre-loaded, washed human platelets was correlated with the levels of [Ca2+]i, using platelets loaded with quin 2. Concentrations of OAG (62-125μM) and diCg (15-30μM), which have previously been shown to be fully effective at activating protein kinase C, failed to significantly enhance [14C]-5HT secretion in combination with ADP (10μM), adrenaline (10μM) or PAF (0.2μM) although they potentiated platelet aggregation, when added 10-30 sec after these agonists to I-PL. eg ADP-0%, 30jiM diCg-9.8%, ADP+diCg-11.9%, 5HT release (p>O.05). In contrast, a low concentration of U46619 (0.2μM), that induced no aggregation, [14C]-5HT secretion or rise in [Ca2+]i levels on its own, was able to synergize strongly at potentiating secretion in combination with all three weak agonists examined, as well as in combination with OAG and diCg (U46619-0%, ADP+U46619-20.4%, U46619+30μM diC8-48% 5HT release) . The greater effectiveness of U46619 at potentiating secretion in combination with the weak agonists was not related to different degrees of [Ca2+]i mobilisation, as ADP and PAF-induced rise in [Ca2+]i occurred to a similar degree in the presence of U46619 and diCg. At a higher concentration of U46619 (0.6μM), which was maximally effective at inducing secretion and elevating [Ca2+]i levels on its own, addition of the weak agonists or OAG or diCg, along with U46619, resulted in a further enhancement of secretion which was independent of changes in [Ca2+]i levels. The results demonstrate that U46619 but not OAG or diCg, is able to fully restore weak agonist-induced secretion in indomethacin-treated platelets, suggesting that the actions of endogenously formed PG endoperoxides/TxA2 cannot be substituted by DAG and raised [Ca2+]i levels and, may be mediated via a mechanism additional to that involving these mediators.
EFFECT OF PROTEIN KINASE C (PrkC) ACTIVATORS ON AGONIST-INDUCED ARACHIDONATE RELEASE IN HUMAN PLATELETS
Authors: Krishnamurthi S , Joseph S K , Kakkar V V
Published: August 2018

We have examined the effect of 1,2 dioctanoylglycerol (diCg) and phorbol 12-myristate 13-acetate (PMA), both activators of PrkC, on arachidonic acid (AA) release induced by thrombin, collagen and the Ca2+ ionophore, ionomycin, and correlated this with the levels of [Ca2+]i in quin 2-loaded platelets. Experiments were carried out using phenidone (200μM, a combined lipoxygenase and cyclooxygenase inhibitor)-treated washed, human platelets pre-labelled with [3H]-AA or quin 2 and, sub-maximal concentrations of thrombin, collagen and ionomycin. Incubation of platelets with diCg (30 or60μM) or PMA (16nM) over a 15 min period, resulted in no significant release of AA or rise in [Ca2+]i levels compared to that in resting platelets. However, thrombin (0.2U/ml) induced a 6-8% release of [3H]-AA as well as a rise in [Ca2+]i equalling 1μM and, addition of diCg (30-60μM) or PMA (16nM) 10 sec-5 min before or 10-20 sec after thrombin, resulted in a significant reduction (20-40%) of both responses. On the other hand, [3H]-AA release in response to collagen (20μg/ml) and ionomycin (6μM) were significantly potentiated by 1.2-1.9 fold and 1.5-3.0 fold over control respectively, by pre-or post-agonist additions of diCg or PMA.As collagen induced no significant rise in [Ca2+]i levels, the differential effects of diCg and PMA on collagen versus thrombin-induced AA release may be related to inhibitory effects on [Ca2+]i mobilisation induced by the latter. Dose-response curves of ionomycin-induced [Ca2+]i mobilisation and AA release revealed that potentiation of the latter by 10 sec pre-incubations of diCg or PMA, only occurred at [Ca2+]i levels> 1μM, with diCg and PMA not affecting the ionomycin-induced rise in [Ca2+]i. The results suggest that the effect of PrkC activators, on agonist-induced AA release, may depend firstly, on whether AA release is dependent on agonist-induced [Ca2+]i mobilisation and secondly, on whether the latter is inhibited by PrkC activation. Thus, the hypothesis regarding a stimulatory role for PrkC in AA release via lipocortin (Touqui et al, Nature 321, pl77, 1986) needs to be re-assessed in the light of these agonist-related mechanistic differences.
THE SYNERGISM BETWEEN PROTEIN KINASE C ACTIVATION AND CALCIUM MOBILIZATION IN PLATELET ACTIVATION
Authors: Yatomi Y , Higashihara M , Tanabe A , Ohashi T V , Kariya T , Kume S
Published: August 2018

It has been shown that Ca2+ mobilization and protein kinase C (C kinase) activation act synergistically to elicit the full physiological response in several systems. That was first demonstrated for the release of serotonin from platelets. To clarify whether or not such synergism can be extended to aggregation and contrast platelet functions with cytosolic free Ca2+ levels( [Ca2+]I), we observed platelet aggregation, serotonin release and [Ca2+], simultaneously by using platelets loaded with 14C-serotonin and Ca2+-sensitive photoprotein aequorin. To induce C kinase activation and Cafmobilization independently, we used the exogenous addition of 12-0-tetradecanoylphorbol 13-acetate(TPA) for the former and a Ca+ionophore, ionomycin, for the latter. Platelet-rich plasma was obtained from healthy human blood and was preincuvated with 1μM14C-serotonin for 45min. Then aequorin was introduced into platelets by incubation with 10mM EGTA and 5mM ATP as described by Johnson et al. Finally, gel-filtered platelets(GEP) were prepared and the count was adjusted to 3×108/ml with Hepes-Tyrode’s buffer containing 1mM Ca2+. GFP were preincubated with aspirin (100μ/ml) for 5 minutes, and purified human fibrinogen(500μg/ml) was added shortly before agonists. Aggregation and aequorin signal were measured by using Chronolog P.I.C.A. The synergistic effects of TPA(50-200nM) and ionomycin(50-200nM) were evident both in aggregation and serotonin release. GFP stimulated by 50nM TPA or 50nM ionomycin alone did not aggregate and released little serotonin. But when they were added together, marked aggregation and release reaction were observed. Aggregation was partially affected by apyrase(2mg/ml). Ionomycin caused a rapid increase in [Ca2+]; in a dose-dependent manner, but TPA (below 200nM) did not in this system. When ionomycin and TPA were added together, [Ca2+]; was dependent on the concentration of the former. It is concluded that synergism between Ca2+ mobilization and C kinase activation is observed for platelet aggregation as well as release reaction.
SYNERGISM OF Gi-DISSOCIATION AND PROTEIN KINASE C STIMULATION IN PLATELET ACTIVATION
Authors: Siess W , Lapetina E G
Published: August 2018

Epinephrine or UK 14304 (a specific (α2-adrenoceptor agonist) synergizes with phorbol esters (phorbol 12,13-dibutyrate, PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol, DiC8) to induce aggregation and ATP-secretion of platelets. The effect on aggregation is more pronounced than on secretion, and it is observed in aspirinized platelet-rich plasma or suspensions of washed platelets containing ADP-scavengers. No prior shape change is found. In the presence of epinephrine, DiCg induces reversible aggregation and PdBu evokes irreversible aggregation that correlates with the effects, on protein phosphorylation. Epinephrine and UK 14304 neither induce nor enhance the phosphorylation of myosin light chain (20kDa), the substrate of protein kinase C (47kDa), or a 38kDa protein evoked by DiCg) or PdBu. Epinephrine does not cause, stimulation of phospholipase C as reflected by the production of inositol mono-, bis- and tris-phosphate or phosphatidic acid. Even under conditions of maximal aggregation induced by epinephrine plus PdBu, formation of 32p-phOSphatidic acid is not observed. The synergistic action of epinephrine and PdBu does not depend on extracellular Ca2+. Primary aggregation induced by epinephrine, but not platelet aggregation induced by PdBu plus epinephrine, is inhibited by high intracellular concentrations of the calcium chelator quin2. Prostacyclin prevents platelet aggregation but does not affect protein phosphorylation induced by PdBu plus epinephrine.

The experiments indicate that α2-adrenoceptor agonists may induce primary aggregation by a mechanism involving release of membrane-bound Ca2+. The synergism with protein kinase C is, however, caused by a mechanism that occurs distally to protein phosphorylation and is not related to Phospholipase C activation and Ca2+-fluxes across the Dlasma membrane or in the cvtosol. Evidence is presented suoportina the view that this mechanism miqht be related to the dissociation of Gi caused by α2-adrenoceptor activation.
POSSIBLE RELATIONSHIP BETWEEN THE 23-kDa PHOSPHOPROTEIN AND THE IP3 -INDUCED Ca2+RELEASE IN HUMAN PLATELETS
Authors: Enouf J , Bredoux R , Giraud A , Bourdeau N , Levy-Toledano S
Published: August 2018

The regulation of Ca2+ concentration in human platelets involves intracellular membranes i.e. dense tubular system (DTS). Agonist-induced platelet activation generates inositol 1,4,5 trisphosphate (IP3) which is responsible for Ca2+ mobilization from DTS. However, its mechanism of action is still unknown. cAMP has been shown to regulate Ca2+ transport by isolated membrane vesicles. This effect was correlated with the phosphorylation of a 23 kDa protein. We investigated whether this phosphorylation could play a role in the mechanism of IP3-induced Ca release.

We isolated a membrane fraction enriched in intracellular membranes, which actively sequesters Ca2++. The Ca2+ uptake was mediated by a characterized (Ca2+ + Mg2+)-ATPase of a molecular weight 120 kDa. As well, the characterization of the 23-kDa protein phosphorylation by the catalytic subunit of the cAMP dependent protein kinase (C. Sub.) has been achieved.
IP^-induced Ca release was tested on our membrane preparations. The transient effect was maximal at one minute and a dose-response curve was obtained.

The cAMP dependent phosphorylation of the 23-kDa protein increased the Ca2+ liberation induced by IP by two fold whatever the IP3 concentration. The addition on the protein kinase inhibitor inhibited the IP3 -induced Ca2+ release.

The effect of IP3 on the cAMP-mediated phosphorylation of the 23-kDa protein has been examinated.A dose dependent stimu-ulation of the 23-kDa protein phosphorylation in the presence of C. Sub. was initiated by IP3. The maximal effect was observed after 1-2 min and obtained with an IP3 concentration similar to that producing the maximal calcium release. The stimulation of the phosphorylation by IP3 was detected in the absence of Ca2+ and in the presence of phosphatase inhibitors.

Therefore, we suggest a possible correlation between cAMP dependent phosphorylation of the 23-kDa protein and the IP3-induced Ca2+ release in human platelet membrane vesicles.