bluuebunny/crossref_metadata_2025 huggingface dataset:
KINETIC ANALYSES OF THE ACTIVATION OF GLU- OR LYS-PLASMINOGEN BY STREPTOKINASE IN THE PRESENCE OF FIBRIN OR ITS DEGRADATION PRODUCTS
Authors: Takada Y , Sugawara Y , Makino Y , Takada A
Published: August 2018

Human plasminogen was activated better by streptokinase (SK) in the presence of fibrin or its degradation products(potentiating agents). Such potentiating agents must be mixed with SK and plasminogen simultaneously. The addition of potentiating agents to the mixture of SK and plasminogen did not result in any enhancement of the activator activity of SK-plasminogen complex. Since potentiating agents such as fibrin bind to lysine binding sites (LBS) of plasminogen, and SK was known to bind to light chain side of plasminogen, it appears that a trimolecular complex of SK, plasminogen and a potentiating agent is a better enzyme than a complex of SK and plasminogen. The effectiveness of the potentiation was in the order of fibrin> SK-potentiator(early FgDP)> fibrinogen >fragment D >fragment E, when Glu-plasminogen was activated by SK. When Lys-plasminogen was used, fragment E was more effective than fragment D, thus in the order of fibrin> SK potentiator> fibrinogen>fragment E >fragment D. Such enhancement of the activation of plasminogen by fibrin or potentiating agents is more significant in the presence of lower concentration of SK. Increase in the concentration of SK did not result in much enhancement of the activation of plasminogen. Kinetic analyses indicated that kcat of the trimolecular complex (SK, plasminogen and potentiating agent) was higher than SK-plasminogen complex, without change in Km. These results suggest that a trimolecular complex of SK, plasminogen and potentiating agent converts plasminogen to plasmin more effectively than a dimole-cular complex of SK and plasminogen. Considering the fact that the plasma concentrations of thrombolytic agents such as SK or UK were low even after drip infusion of their large dose Intravenously, the potentiation of the activation of plasminogen by SK in the presence of fibrin maybe significant in the mechanisms of thrombolytic therapy.
MONOCLONAL ANTIBODIES AGAINST FOUR DIFFERENT EPITOPES OF PLASMINOGEN EXHIBIT DIFFERENT EFFECTS ON PLASMINOGEN ACTIVATION KINETICS
Authors: Hattey E , Wojta J , Binder B R
Published: August 2018

Monoclonal antibodies against four different epitopes of plasminogen (PG) were obtained and characterized for their binding towards PG using immunoblotting of native, degraded, elastase cleaved PG and alpha-2-antiplasmin complexed plasmin. One antibody (MPW1PG) recognized exclusively Glu-forms of PG and plasmin, a second antibody (MPW2PG) was bound to the kringle 1-3 fragment, one antibody (MPW7PG) recognized the kringle 4 fragment and the fourth antibody (MPW4PG) miniplasminogen. All antibodies exhibited affinities towards PG in the same order of magnitude. From these antibodies only MPW4PG inhibited plasmin action (competitive inhibitor, Ki = 4nM) and after complex formation of plasmin with alpha-2-antiplasmin the complex was not recognized. All other antibodies exhibited binding also to the respective alpha-2-antiplasmin complexed forms. The three antibodies directed against the different epitopes of the heavy chain of plasmin stimulated activation of Glu-PG by urokinase (UK), thereby mimicking the effect of lysine (KM unchanged, kcat increased) but not activation of Lys-PG. When single chain tPA was used instead of UK and PG activation was studied in the absence and presence of CNBr fragments of fibrinogen none of the three antibodies (MPW1PG, MPW2PG, MPW7PG) interfered significantly with the stimulating effect of fibrin, but the lag phase, until maximal fibrin stimulated plasmin formation occured, was reduced. In the absence of fibrin all three antibodies increased the rate of Glu-PG activation by tPA also increasing kcat values while addition of lysine rather inhibited PG activation by tPA and completely abolished the fibrin effect. These results are consistent with the plasmin induced decrease in lag time of the fibrin effect found for these antibodies. From these data it can be concluded that binding of an antibody to one of the three described domains in the heavy chain induces conformational changes in PG leading to an increased rate of activation by both activators. Interestingly, binding of these antibodies did not interfere with the fibrin effect and is different from the effect of lysine.
KINETIC ANALYSES OF THE ACTIVATION OF GLU-PLASMINOGEN BY TISSUE PLASMINOGEN ACTIVATOR IN THE PRESENCE OF FIBRIN, FIBRINOGEN, FRAGMENTS D AND E
Authors: Takada A , Sugawara Y , Watahiki Y , Takada Y
Published: August 2018

Kinetic analyses were made for the activation of Glu-plg (Glu-plasminogen) by tissue plasminogen activator (t-PA: two chain t-PA obtained from Bowes melanoma cell line) in the presence of fibrin, fibrinogen, fragment D or E. Glu-plg was activated by t-PA in the presence of various concentrations of fibrinogen, fibrin, fragment D or E, and the activation rate was measured by the hydrolysis of S-2251 with plasmin generated. Straight Line-weaver-Burk plots were obtained by changing the concentration of Glu-plg. In the absence of fibrin, t-PA activated Glu-plg with Km of 1.11 μM, kcat of 0.005 sec-1, kcat/Km of 0.0045 μM-1,sec-1. The presence of catalytic amounts (0.0001 μM) of fibrin resulted in 11.4 fold decrease in Km, thus 15.6 fold increase in kcat/Km. The presence of larger amounts of fibrin resulted in increase in kcat and decrease in Km, which resulted in 142 fold increase in kcat/Km. There seems to be two phases in the enhancement of the activation of Glu-plg by t-PA, one induced in the presence of catalytic amounts of fibrin, and the other induced in the presence of higher concentration of fibrin (up to 0.05/0.2 of the molar ratio of fibrin to plasminogen). When effects of fragment D or E were examined for the enhanced activation of Glu-plg by t-PA, the presence of increasing amounts of E resulted in increase in kcat. Presence of D resulted in increase in kcat and decrease in Km. Since plasminogen was shown to bind to both D and E domains of fibrin, and t-PA was shown to bind to D domain, we hypothesize that the role of D in the activation of Glu-plg by t-PA was to provide a site to t-PA and plasminogen to form a ternary complex, which facillitates the activation of plasminogen by t-PA, and that the role of E was to provide a site to bind to plasminogen, where t-PA activates plasminogen more effectively. The results suggest that roles of D and E domains of fibrin may be different in the enhanced activation of plasminogen by t-PA.
EFFECT OF pH DURING PREPARATION OF EUGLOBULIN PRECIPITATES ON t-PA OR UK ASSAY IN PLASMA
Authors: Kunitomi O , Kakishita E , Nagai K , Matsuo O
Published: August 2018

A more sensitive method for the commonly used euglobulin method is proposed for examining plasminogen activators (PAs) in blood, since little attention has been paid to the conditions of production of euglobulin precipitates, especially the pH during their preparation. In the present study, the effect of pH on measurement of the activity of tissue-type plasminogen activator (t-PA) or urokinase (UK) in plasma was investigated and compared by enzymography, bio-immunoassay (BIA) and the fibrin plate method. The antigenicity of PAs was observed as follows. Polyclonal anti-t-PA or UK rabbit IgG previously purified with aprotinin-Sepharose to remove protease activity in the IgG fraction (final concentration, 0.375 mg/ml of 0.01% Triton X-100) was reacted with euglobulin precipitate at 4°C for 1 hr. After incubation, enzymography was preformed, and the residual fibrinolytic activity was measured qualitatively. Euglobulin precipitates were prepared by dilution (1:20) of fresh venous occlusion plasma obtained from healthy volunteers (n = 5) with ice cold, distilled water, and adjusted to pH 4.5 - 7.5 with 0.1% (v/v) acetic acid. The euglobulin precipitates were collected by centrifuge at 4°C, and dissolved in Veronal buffer (0.1 M NaCl, 0.05 M barbital Na, pH 7.75) to the original volume. The PA activities in these solutions were examined by the above three methods. The fibrinolytic activities between pH 5.0 and 6.8 were found to be almost the same (no statistical difference) by the fibrin plate method. However, a stronger t-PA activity as determined from the molecular weight as well as antigenicity, was detected at pH 6.7 by enzymography and BIA. On the other hand, a stronger UK activity as determined from the molecular weight and antigenicity was recognized at pH 5.6 by enzymography. The present results suggest, therefore, that it is necessary to consider the effect of pH during the preparation of euglobulin precipitates for the measurement of t-PA or UK assay in plasma. The suitable pH value for t-PA and UK assays in plasma is 6.7 and 5.6, respectively.
RELEVANCE OF FREE t-PA ASSAY FOLLOWING VENOUS OCCLUSION IN PATIENTS WITH VENOUS THROMBOEMBOLIC DISEASE
Authors: Alessi M C , Juhan-Vague I , Valadier J , Philip-Joet C , Holvoet P , Collen D
Published: August 2018

Out of 100 consecutive patients with a history of venous thromboembolism, 80 were subjected to 10 min venous occlusion (VO). Blood samples were collected on citrate, immediately cooled on ice, centrifuged and frozen. Euglobulin fibrinolytic activity (EFA) was measured on fibrin plates, plasminogen activator inhibitor (PAI) activity according to Verheijen et al., and total t-PA antigen with an ELISA. In addition, free t-PA was assayed in plasma collected on the monoclonal antibody MA-2G6, which is directed against the active site of t-PA.

The assays allowed to differentiate three groups of patients. Good responders (GR) to VO with at least a doubling of the EFA after venous occlusion. Poor responders (PR) with less than a two-fold increase in EFA could be classified either as having a high PAI level resulting in rapid inhibition of t-PA (PR-I) or as having a deficient release of t-PA from the vessel wall (PR-II).

It is concluded that patients with a history of venous thromboembolism may have an abnormal fibrinolytic response to venous occlusion which is due either to an increased inhibitor level or to a deficient t-PA release. Both abnormalities are recognized by the free t-PA assay.
ASPIRIN DOES NOT INHIBIT FIBRINOLYTIC ACTIVITY IN TIA PATIENTS AFTER VENOUS OCCLUSION
Authors: Carriero M R , Pintucci G , Castagnoli M N , Colombo R , Lombardi B , Mussoni L , de Gaetano G
Published: August 2018

We have recently shown that In normal subjects aspirin (1,300 mg) and Indobufen (400 mg), a new cyclo-oxygenase Inhibitor structurally unrelated to salycilate, lower the fibrinolytic activity, without modifying t-PA antigen levels, after venous occlusion (VO). The aim of this study was to investigate whether aspirin reduces fibrinolytic response to VO also In patients with TIA. These patients were selected In view of controlled clinical trials showing reduction of TIA recurrency and stroke by treatment with high dose aspirin. Six males (56-65 yrs old), with previous TIA (< 1 year) were selected; the presence of diffuse atherothrombotlc lesions was demonstrated by doppler sonography and angiography. All patients were given, ten days apart, aspirin (600 mg daily x 2) or Indobufen (200 mg daily x 2) following a randomized cross-over single blind scheme. In all patients 10 minutes VO applied before any drug administration, Induced activation of the fibrinolytic system as assessed by euglobulin lysis area on fibrin plates (from 226±47 to 643±57 mm2), t-PA antigen (from 13.8± 1.0 to 40.9±3.1 ng/ml) and PA-1 activity (from 39.5+5.0 to 14.8±1.6 AU/ml). Neither aspirin nor Indobufen Ingestion resulted In any Inhibitory effect on fibrinolytic response to VO while both drugs suppressed serum thromboxane 02 generation by more than 98%. In conclusion high dose aspirin and Indobufen do not Impair the fibrinolytic potential In TIA patients with atherothrombotlc lesions. The reasons for the different behaviour of patients In respect to young healthy volunteers remain to be established.
EFFECT OF VENOUS STASIS ON THE RELEASE OF VON WILLEERAND FACTOR ANTIGEN (VWF:AG) AND PLASMINOGEN ACTIVATOR (PA) IN PATIENTS WITH THROMBOEMBOLISM
Authors: Uchiyama T , Matsumoto M , Narahara N , Tanaka H , Kobayashi N , Maekawa T
Published: August 2018

Plasma levels of vWF:Ag and PA (both activity and antigen) present in the venous blood were studied in 114 patients with arterial thromboembolic disease and 30 age matched healthy individuals. In 29 cases of the patient group (patients) and 7 cases of the control group (controls), turnover of intravenously injected 1-125-fibrinogen was studied. Venous blood was obtained from the antecubital vein of subjects before and after 5 minutes0' of venous occlusion. vWF:Ag was determined by electroimmunodiffusion (Laurell0's method). PA activity was measured by the method of Campbell et al, and PA antigen was assayed by ELISA kit purchased from BioPool Co. And the following results were obtained : 1) Mean plasma level of vWF:Ag was significantly higher (p<0.001) and mean plasma level of PA activity was significantly lower (p<0.05) in patients than in controls both before and after the venous occlusion. 2) Mean plasma level of PA antigen was significantly higher (p<0.01) in patients (mean+SD; 4.05j±1.58 ng/ml) than in controls (2.95±1.11 ng/ml) before the venous occlusion. The mean specific acitivity of PA was significantly lower (p<0.01) in patients than in controls both before and after the venous occlusion. 3) Plasma half life (T/2) of fibrinogen was significantly shorter (p<0.001) and catabolic flux (J3X) of fibrinogen was significantly higher (p<0.001) in patients tahn in controls. 4) Significant relationship was observed between T/2 of fibrinogen and plasma levels of vWF:Ag before and after the venous occlusion, PA activities after the occlusion, and levels of PA antigen before the occlusion. 5) Significant relationship was also observed between J3X of fibrinogen and plasma levels of vWF:Ag before and after the verous occlusion, PA activities after the occlusion, and levels of PA antigen before the occlusion. These results suggests that the changes in endothelial cell function might be a common mechanism responsible for the abnormal findings in plasma levels of both vWF:Ag and PA and for the acceleration of fibrinogen metabolism.
LEUCOCYTES AND PLATELETS DEPOSITION CN ENDOTHELIAL SURFACE INDUCED BY PARTIAL VENOUS OCCLUSION.AN ULTRASTRUCTURAL STUDY IN THE DOG
Authors: Andrade J Aguiar , Resende C , Monteiro A C , Adolfo A , Pina Cabral J M
Published: August 2018

After partial venous occlusion of dog's femoral vein until 60% of its initial cross section for 90 min. we have observed a decrease in plasminogen activator content (t-PA) in the pre-occlusion segment and an increase of prostacyclin like activity (PLA) in the post -occlusion segment (in press).

In this work and in order to try to find an ultra-structural basis for these findings, we carried out observations of the constricted segments with transmission and scanning E.M. (n=10).

We observed that : -1) endothelial cells were maintained in place, with minimal ultrastructural changes, namely a small increase in vaccuolization ;-2) there was massive subendothelial deposition of leucocytes, predominantly polimorphonuclears, which permeate internal elastic lamina and were also found in subjacent structures; -3) platelets were rarely seen and platelets agregates were never found. Autologous Indium labelled platelets injection confirmed "in vivo" these findnigs (n=10).

We conclude that in partial venous occlusion, platelet deposition and endothelial cell descamation are not initiating factors in changes observed at t-PA and PLA vessel wall levels, although the role of white blood cells will need further investigation.
ISOLATION OF THE GENOMIC REGION CODING FOR TYPE-1 PLASMINOGEN ACTIVATOR INHIBITOR
Authors: Strandberg L , Lawrence D , Ny T
Published: August 2018

The type-1 Plasminogen Activator Inhibitor (PAI-1) has recently been identified as a member of the Serine Protease Inhibitor family (SERPINS). This family of proteins contain many serine protease inhibitors but also functionally unrelated proteins like ovalbumin and anginotensinogen. PAI-1 inhibits both u-PA and t-PA and might therefore be an important regulator of the fibrinolytic system.

In order to study the evolution of the Serpin family as well as PAI-1 gene expression we have isolated the genomic region carrying the PAI-1 gene. A cDNA sequence for PAI-1 was used as probe to screen a human genomic library. When 2 million independent phages were screened, 13 positive clones were isolated. Characterisation of these clones showed that they could be divided into 3 overlapping groups covering a genomic region of approximately 30 kb. The gene was localized and characterized by restriction enzyme analysis, southern blotting using cDNA and oligomer probes, and DNA sequencing.
MEASUREMENT OF PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI-1) IN PLASMA WITH AN ELISA BASED ON TWO MURINE MONOCLONAL ANTIBODIES
Authors: Declerck P J , Alessi M C , Verstreken M , Kruithof E K O , Juhan-Vague I , Collen D
Published: August 2018

An enzyme-linked immunosorbent assay (ELISA) for quantitation of plasminogen activator inhibitor 1 (PAI-1) was developed, based on two murine monoclonal antibodies (MA-7D4B7 and MA-7F5), raised against purified PAI-1 from HT-1080 fibrosarcoma cells, which react with non-overlapping epitopes. MA-7D4B7 was coated on microtiter plates and bound PAI-1 antigen was quantitated with MA-7F5 conjugated with horseradish peroxidase.

In normal plasma, collected on citrate at pH 7.4, the PAI-1 level is 27 ± 16 ng/ml (mean ± SD, n=ll), with a corresponding value of 19 ± 11 ng/ml (n=12) in plasma collected on acid citrate pH 4.5, which inhibits the release of PAI-1 from platelets. The lower limit of the assay in plasma is 2 ng/ml; the intra-assay, inter-assay and inter-dilution coefficients of variation are 5.2%, 8.0% and 7.1% respectively.

This ELISA was used to measure PAI-1 levels in plasma (collected on citrate, pH 7.4) of non-pregnant women and of women at different stages of pregnancy. A progressive increase is observed : before: 20±9 ng/ml, n=7; first trimester: 25 ± 12 ng/ml, n=5; second trimester: 40 ± 25 ng/ml, n=ll; third trimester: 98 ± 46 ng/ml, n=13. A correlation coefficient of 0.70 is found between the duration of pregnancy and the PAI-1 level.

Preliminary data indicate that the PAI-1 antigen level is increased in several disease states, including myocardial infarction and deep vein thrombosis.

Thus, this newly developed ELISA allows a direct measurement of the fast-acting inhibitor of plasminogen activator in plasma. Application of this assay to plasma of non-pregnant and pregnant women substantiates previous results obtained with the use of functional assays. In order to quantitate PAI-1 antigen circulating in plasma, blood should be collected under conditions that prevent platelet stimulation.
A SIMPLE AND RAPID PLASMINOGEN ACTIVATOR INHIBITOR (PAI) ASSAY IN PLASMA, USING A UNIQUE CONCENTRATION OF TISSUE PLASMINOGEN ACTIVATOR (tPA) AND PAI-DEPLETED PLASMA
Authors: Contant g , Anglescano E , Martinoli J L
Published: August 2018

Interferences of plasmin inhibitors are a main drawback in the functional assay of PAI in plasma. To overcome them, an acidification step, prior to residual tPA assay has been frequently used.

A simple chromogenic method, based on the inhibition of purified tPA by plasma prediluted in PAI-depleted plasma, is described. The residual tPA was subsequently measured in the presence of fibrinogen fragments used as Stimulator, plasminogen and chromogenic substrate (CBS 30.41). The assay can discriminate between PAI and plasmin inhibitors since the concentration of these proteins remains constant because brought in excess by the PAI-depleted plasma. Parallel dose-response curve were obtained when PAI-depleted plasma was used instead of buffer, for the preparation of the reference curve and for the dilution of samples. The residual tPA activity measured was directly proportional to the PAI activity of the sample using a single concentration of tPA. Samples containing 0 to 30 IU/ml of PAI could be measured using a 1:40 dilution.

Values for 27 normal volunteers at rest were 5.1 IU/ml (range : 0 to 11.8 IU/ml). PAI activity was decreased in afternoon samples (4.6 IU/ml) compared to those of the morning (5.8 IU/ml). After stasis, when a concomitant release of tPA was observed, the values obtained (3.1 IU/ml) were significantly lower than the values obtained before (4.1 IU/ml).

The activity measured is probably related to PAI-1 (endothelial ceil type PAI), but PAI-2 related activity (placental PAI) can be also estimated since pregnancy plasma showed high levels of inhibitor (mean : 15.1 IU/ml, range : 9.5 to 28.1 IU/rni).

The present method is simple and rapid to perform. It can be routinely used in the screening of various thrombotic disorders.
DETERMINATION OF PA INHIBITOR 1 (PA INHIBITOR ACTIVITY AND PA INHIBITOR 1 ANTIGEN) IN PLASMA BEFORE AND AFTER VENOUS OCCLUSION
Authors: Juhan-Vague I , Valadier J , Alessi M C , Ansaldi J , Ailluad M F , Kruithf EK O
Published: August 2018

PA Inhibitor activity (PAIact-Verheijen’s method-U/ml) and PA Inhibitor 1 antigen (PAIlAg- Kruithof’s radioimmunoassay -ng/ml) were evaluated, on blood samples before (B) and after (A) venous occlusion (VO) (10 min) in order to analyse, in B-VO samples, the ratio (R=PAIlAg/PAIact) ' between the immunological and enzymatic material, and the change of PAIlAg levels after VO.

The B-VO values (m ± SD) were determined from 111 plasmas : -86 with normal ( 12U/ml) PAIact levels = 3.7 ± 2.5 ; PAIlAg = 17.4 ± 10.1 ; R = 7.1 ± 6.2 (range 1.5 - 24 )-25 with high PAIact levels (15 post surgery, 10 obese patients)= 30.9± 19.3; PAIlAg = 75.3 ± 44.7 ; R = 2.7 ± 1.5 (range 1.3- 4.4). The correlation between the 2 dosages was r = 0.82 (p 0.01). In the 2 kinds of patients with high PAIact levels, a parallel high PAIlAg level was found. As the range of the ratio R was very large, patients were divided in 2 Groups : GrI : Normal R ( 7), n=81 (normal PAIact : n=57 ; high PAIact : n=24) and GrII : high R, n=30. The results for PAIact/PAIlAg/R were = GrI :12.6± 16.3/ 31.5±34.4/ 3.2 ± 1.5. GrII : 2.6±4.1/ 27.9 ±30.1/ 13.8±6.1. In GrII the platelet origin of inactive PAIlAg from in vitro activation of platelets could be demonstrated (high level of BTG lug/ml) in 50 % of cases. No in vitro platelet activation could be demonstrated in GrI. These results point out the necessity of a good preparation of the plasma (mainly 0-4°) for PAIlAg determination.

The B and A-VO values were analysed from 60 subjects. Plasmas with in vitro activation of platelet determined by BTG levels had been discarded. No platelet activation occured with the VO. The results were not corrected with the PCV. PAIlAg B-VO/A-VO = 19.2 ± 15.1/ 26.9 ± 18.3. 36 subjects (60 % of total) had an increase in PAIlAg A-VO 20 % (mean increase = 62 %) . There was no correlation between basal values of PAIlAg and the increase after VO, and between PAIlAg increase and t-PA-Ag release. It is concluded that a weak increase of PAIlAg may occur after VO ; this increase is not parallel with t-PA release. The physiopathological significance of this increase of PAIlAg as yet to be evaluated.
DIFFERENTIAL EFFECT OF TEMPORARY Ca2+ DEPRIVATION UPON ADP-AND COLLAGEN-INDUCED PLATELET AGGREGATION IN HUMAN WHOLE BLOOD
Authors: Watts I S , Keery R J , Lumley P
Published: August 2018

In platelets exposed to chelating agents at 37°C and recalcified, aggregation to ADP, adrenaline, collagen and thrombin were either abolished or markedly reduced (1). This phenomenon has been suggested to be caused by disruption, by Ca2+ deprivation, of the fibrinogen receptor (glycoprotein lib Ilia complex) on the surface of the platelet (2). This effect was dependent on the exposure time of Ca2+ chelators and was reduced at lower temperatures. We have investigated the effect of exposure of human platelets to EGTA upon aggregation to a range of agonists at both 37 and 25°C. EGTA (5 mM) was incubated with citrated (12.9 mM) whole blood for either 0, 1, 5, 15 or 30 min. One minute following recalcification (5 mM CaCl2), full aggregation concentration-response curves were constructed to ADP and adrenaline (0.1 to 3 μM), PAF (3 to 100 nM), collagen (0.1 to 4 μg/ml) and U46619 (0.03 to 1 μM), using a platelet counting technique (3). At 25°C, EGTA produced little or no significant loss of sensitivity to any agonist. At 37°C marked rightward shifts of the ADP, adrenaline and PAF aggregation curves occurred which were related to the time of incubation with EGTA (e.g. ADP concentration ratios (CR) of 2.0 (0.8-4.8), 4.7 (2.1-10.7), › 186.6 (135.2-257.6) (95% confidence intervals n=4) obtained at 1, 5 and 15 min respectively). Following 30 min incubation aggregation to all three agonists was abolished (up to ADP 300 pM; adrenaline 100 μM; PAF 10 μM). In contrast, whilst collagen and U46619 concentration-effect curves were displaced to the right following 30 minutes exposure to EGTA (CR =13.6 (6.1-30.4) and 9.0 (4.3-18.7) respectively, n=4) full aggregation curves could still be established. Furthermore, a 60 min incubation with EGTA caused little further effect. Our findings suggest platelet agonists such as collagen and U46619, but not ADP, adrenaline and PAF, can evoke expression of a population of fibrinogen receptor sites involved in platelet aggregation that are inaccessible to EGTA.

(1) Zucker, MB & Grant, RA (1978). Blood, 52, 505. (2)Shattil, SJ et al. (1985). Blood, 66, 92. (3) Lumley, P & Humphrey, PPA (1981). J. Pharm. Methods, 6, 153.
PHOSPHOLIPASE A2 ACTIVATION BY A MECHANISM SEPARATE TO THAT RESPONSIBLE FOR PHOSPHOLIPASE C STIMULATION IN ALPHA-THROMBIN-STIMULATED HUMAN PLATELETS
Authors: Crouch Michael F , Lapetina Eduardo G
Published: August 2018

The ability of cell surface receptor occupation to increase the activity of phospholipase A2 has been thought to be due to the prior activation of phospholipase C and an increase in the intracellular Ca2+ concentration. However, recent evidence from our and other laboratories has suggested that this may not be the case, but rather stimulation of phospholipase A2 may be under the control of separate receptor-activated events. We have investigated this further by comparing the ability of prostacyclin (PGI2) and epinephrine to alter platelet responses to thrombin and examining the resulting phospholipase A2 activities.

Alpha-thrombin stimulated aggregation of human platelets, the formation of inositol phosphates and phosphatidic acid, mobilizaton of Ca2+ from internal stores and Ca2+ influx, protein phosporylation (47 kDa and 20 kDa) and arachidonic acid (AA) release. Each of these responses was partially inhibited by prostacyclin (PGI2) except that of AA release, which was abolished. In combination with epinephrine and PGI2, alpha-thrombin-induced aggregation, phosphatidic acid formation and protein phosphorylation were restored, but the release of AA only reached 50% of its control value. Epinephrine alone had no effect on any of these responses, either in the presence or absence of PGI2. Thus, alpha-thrombin-induced activation of phospholipase A2 is more sensitive to the effects of PGI2 than is phospholipase C, and supports the possibility that there are distinct control mechanisms for receptor activation of these enzymes. We are presently examining the role of Gs in the inhibition by PGI2 of platelet phospholipase A2 and of Gi in the thrombin stimulation of this enzyme
THROMBIN-INDUCED STIMULATION OF HUMAN PLATELETS LEADS TO AN INCREASE IN CYTOPLASMIC CALCIUM
Authors: Davies T A , Drotts D , Weil G , Simons E R
Published: August 2018

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are detected with indo 1. They are extremely rapid and are maximimal by 1015 seconds. Suspension studies, which reflect an average value over 3 × 107 cells/ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium. However, flow cytometry indicates that subpopulations of cells are responding differently. The responses of these populations seem to be dependent on thrombin concentration. A maximal overall response is found when more than 50 % of the cells respond. This may explain multiple stimulations of the same suspension of cells at low thrombin doses

Elevated extracellular Ca++ increases the maximal cytoplasmic response and significantly retards its subsequent decrease. When extracellular Ca++ is removed via addition of EGTA just before stimulation, the initial response is halved and the subsequent slow decrease is more marked and returns to lower (near-basal) levels. Intracellular Ca++ can be chelated with s - (0-am inophenoxy) - e thane-N, N, N',N'-tetraacet ic acid (BAPTA), which has a twofold greater affinity far Ca++ than does indo, but which does not fluoresce (at indo sensitive wavelengths) upon binding of Ca++. Cells loaded with both indo and BAPTA exhibit no rise in cytosolic Ca++ or altered membrane potential when stimulated with αthrombin, unlike cells loaded with indo alone. When Ca++ (2 mM) is added back to the extracellular buffer, these cells will take up the Ca++ at a constant rate as seen by a rise in indo fluorescence. When cytosolic Ca levels reach those of the resting platelets loaded with indo alone, these cells recover the αthrombin-indue ed Ca response of control platelets. However, they no longer depolarize partially under these same Ca++ replenished conditions. This implies that some of the Ca++ required for the platelet thrombin response comes from non-replenishable internal stores.
EFFECT OF AGE AND SEX ON THE PLATELET FATTY ACID (FA) COMPOSITION AND IN ITS MODULATION BY PLASMA FA
Authors: Vallés J , Santos M M , Aznar J
Published: August 2018

Age, sex and platelet hyperactivity are factors that condition the development of coronary heart disease (CHD). Platelet function is believed to be influenced by its FA pattern, which can be modulated by the FA composition of plasma lipid fractions. Therefore, it seemed of interest to evaluate the effect of age and sex on platelet FA and ascertain whether the plasma influence on platelet FA is modified by age and sex. The study has been performed in 98 subjects, 49 couples, men (M) and women (W), living together and with the same dietary habits. The subjects were divided into three age groups : G I:16-40, GII:40-60,and G III: 60. Plasma and platelet FA of phospholipids (PL), triglycerides, and free FA fractions were evaluated by gas-chromatography. The results showed scarce differences in platelet FA in relation to sex. With respect to age, the percentages of 18:2 and 20:5 in most platelet lipid fractions both in M and in W decrease with age. More interesting are the differences in correlation found between plasma and platelet FA, particularly in the PL fraction, with age and sex. In this respect an increase in the correlation coefficient was found for 16:0,18:0 and 20:4 and a decrease for 20:5 in middle aged men and postmenopausic women (Table). The similarity between these two groups of subjects may have a physiopathological meaning, if we take into account that both are more susceptible to CHD and that an increase in 16:0, 18:0 and 20:4 as well as a decrease in 20:5 may condition a platelet hyperfuntion, circumstance that may be more easily produced by plasma influence in those subjects.The results of the present study also confirm that that in general plasma greately influences the platelet content in 18:1, 18:2 and to a lower extent the saturated FA.
The aggregation of bovine platelets is not dependent on thromboxane B2 production
Authors: Gentry P A , Bondy G S
Published: August 2018

Bovine and human platelets appear to be equally sensitive to inhibition by several environmental toxins even though the bovine is not generally recognised as a species prone to thromboembolic and hemorrhagic disease. During the examination of the mechanism of action of the toxins it became necessary to establish parameters for normal bovine platelet function.

Bovine platelets suspended in homologous plasma demonstrate various responses to different agonists. Bovine platelets aggregate effectively and irreversibly to fibrillar form collagen and adenosine-diphosphate (ADP), undergo reversible aggregation in response to platelet activating factor (PAF), exhibit a minimal aggregation response to serotonin (5HT) and arachidonic acid (AA) and are refractory to acid-soluble collagen (calf skin). A synergistic aggregation response is observed with the combination of ADP and 5HT, an additive response with ADP and PAF, while the addition of AA has no effect on the ADP induced aggregation response. A biphasic response has not been observed with any of the agonists or with the combinations of agonists. There is no direct correlation between the maximum aggregation response induced by ADP and PAF and the amount of thromboxane B2 (TXB2) released from the stimulated platelets. Similarly when acetylsalicylic acid (ASA) is added to the platelet suspensions, TBXg release can be completely inhibited without any observable effect on the aggregation response. Platelet aggregation was also unaffected by indomethacin and dipyridamole. Verapamil is a weak inhibitor of ADP induced aggregation but is a potent inhibitor of PAF stimulated platelets. Bovine platelets appear to have some distinct characteristics since they exhibit some responses which are not only different from human platelets but are also different from platelets obtained from other large animals such as the sheep and horse.
DIACYLGLYCEROL AND PHORBOL ESTER REDUCE AEQUORIN-INDICATED [Ca++] ELEVATIONS INDUCED BY THROMBIN OR ADP
Authors: Ware J A , Smith M , Salzman E W
Published: August 2018

Platelet aggregation and secretion induced by phorbol ester (PMA) or diacylglycerol (DAG) are preceded by an increase in [Ca++] that is detected byaequorin, but not by quin2, fura-2, or indo-1, suggesting that these indicatorsreflect different aspects of Ca++ homeostasis, possibly different functional Ca++ pools. Addition of two conventional agonists in subthreold concentrations synergistically enhances the [Ca++] rise and aggregation.However, if PMA or DAG is the first agonist the subsequent quin2-indicated [Ca++] rise after thrombin is reduced.

Whether aequorin-indicated [Ca++] is similarly affected is unknown. We studied gel-filtered platelets loaded with aequorin or a fluorophore and added PMA, DAG, thrombin or ADP, alone or in combination. Either PMA or DAG alone caused a concentration-dependent increase in [Ca++] detectable with aequorin but not with the fluorophores; simultaneous addition of thrombin or ADP with DAG or PMA produced a larger [Ca++] rise than either alone. However, addition of DAG or PMA as a first agonist reduced subsequent aequorin-indicated [Ca++] rises following thrombin or ADP in a concentration and time-dependent manner. Inhibition of ADP or thrombin-induced [Ca++] rise was not always accompanied by inhibition of aggregation or secretion. Combination of subthreshold concentrations of ADP and thrombin produced an enhanced [Ca++] rise and aggregation. However, this synergistic effect was inhibited by preincubation with DAG or PMA. Neither this effect nor DAG-induced [Ca++] rise was inhibited by the protein kinase C inhibitor H-7. In genera^ preincubation of platelets with an agonist enhances Ca rise and aggregation in response to a second agonist; in contrasl protein kinase C activators, which themselves elevate [Ca++] as shown by aequorin, inhibit aequorin-indicated Ca rises after ADP or thrombin, and limit synergism between these two agonists.
1,2-DIOCTANOYLGLYCERIOL (diC8) BUT NOT 1-OLEOYL 2-ACETYLGLYCEROL (OAG) INHIBITS AGONIST-INDUCED PLATELET RESPONSES
Authors: Joseph S K , Krlshnamurthi S , Patel Y , Kakkar V V
Published: August 2018

Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PrkC), has been reported. We have examined the effects of the diacylglycerol (DG) analogues OAG and diCg, both PrkC activators, as well as PMA on intracellular Ca2+ ([Ca2+]i) mobilisation and 5-hydroxytryptamine (5HT) release induced by thrombin (T), collagen (Coll.) and the thromboxane (Tx) mimetic U46619. All studies were performed using washed human platelets pre-labelled with either quin-2 or [14C]-5HT and maximal concentrations of agonists. Neither diCg or PMA elevated [Ca2+]i above resting levels though both agents induced a small (10-15%) secretory response. In response to 0.2U/ml T or 0.6uM U46619, but not 20μg/ml Coll., [Ca2+]i increased from resting levels of lOOnM to 758±108nM and 712±58nM respectively. Addition of diCg (60μM) or PMA (16nM) 10 sec before or after T or U46619 reduced the control responses by 10-15% and 30-80% respectively. In contrast, [14C]-5HT secretion in response to T and Coll. was unaffected by diCg or PMA and in the case of U46619 was potentiated 1.4-1.6 fold over control levels. With longer pre-incubation times (5 min) [Ca2+]i mobilisation was further reduced and an inhibitory effect (10-40%) on agonist-induced secretion was evident. Unlike diCg or PMA, OAG (63μM) had no significant inhibitory effect on agonist-induced [Ca2+]i mobilisation and [14C]-5HT secretion even with long pre-incubation times (5 min). However, like diCg and PMA, OAG potentiated U46619-induced secretion with a 10 sec incubation though it induced no secretion itself. The inability of OAG to inhibit may be related to its lesser potency as a PrkC activator. Over a 10 sec-5 min period OAG caused significantly less 40Kd protein phosphorylation ( < 2-fold increase in [32P]-labelling), compared to diCg and PMA (4-6-fold increase). Our results suggest that diCg may be a better tool as an activator of PrkC and DG mimic than OAG. Further, the time course of inhibition of agonist-induced [Ca2+]i mobilisation by diCg suggests that this effect may constitute a physiologically relevant phenomenon mediated by DG within a single cycle of agonist-induced events.
THROMBOXANE-A2 MEDIATES THE ACTION OF INOSITOL (1.4.5) TRISPHOSPHATE (IP3) IN SAPONIN-PERMEABILISED PLATELETS
Authors: Authi K S , Evenden B J , Hornby E J , Crawford N
Published: August 2018

Inositol trisphosphate (IP3) has now been identified as an important intracellular second messenger that can initiate the release of Ca2+ from intracellular stores in a variety of cells, including platelets. We have studied the effects of IP3 on washed platelets permeabilised with saponin (12-14 μg/mi) which allows penetration into the cell of low M.Wt polar molecules. The permeabilised cells show normal responses to the agonists thrombin and collagen. The addition of IP (1-20 μM) after saponin treatment induces shape change, aggregation and secretion of preloaded [14C] 5HT. Concomitant with these responses, thromboxane is produced in a dose related manner. With 20 μM IP3 thromboxane B2 increases from basal levels of 5-4 ± 3-0 ng/ml to 140 ± 23 ng/ml. Both thromboxane production and the platelet responses induced by IP3 are inhibited by pretreatment with the cyclooxygenase inhibitors, indomethacin (EC50 50 μM) and aspirin (EC50 30 μM). Aggregation and secretion responses to IP3 are also inhibited by thromboxane B2 receptor agonists; EPO 92 (R. Jones, Edinburgh) and AH 23848 (Glaxo Ltd.). If Ca2+ EGTA buffers age used with permeabilised platelets to "lock" the cytosolic [Ca2+] at 0.1 μM, thromboxane production is reduced to the basal level. Intact platelets were labelled with Ca2+ (4h incubation) and after washing, resuspension and saponisation, IP3 induced the release of 20% of the cell associated Ca2+. The release was unaffected by pretreatment with antimycin and oligomycin indicating an gndoplasmic reticulum-lige storage site for the sequestered Ca2+. This IP3 -induced Ca2+ release was also not affected by pretreatment with either cyclooxygenase inhibitors or thromboxane receptor antagonists (EPO 92 and AH 23848). We believe these studies indicate that the action of IP3 in sagonised platelets involves release of intracellularly stored Ca2+, activation of phospholipase A2 and cyclooxygenase, and production of thromboxane A2. The release of thromboxane mediates and/or attenuates platelet responses by acting upon platelet surface receptors.
INSULIN RECEPTORS ARE NOT COUPLED TO THE PHOSPHOINOSITIDE OR ADENYLCYCLASE MESSENGER SYSTEMS IN HUMAN PLATELETS
Authors: Pfliegler G , Arnout J , Kienast J , Wittevrongel K , Vermylen J
Published: August 2018

Insulin receptors have been found not only on its “target Cells” but also on several other cell-types, including human platelets. From studies on adipocytes and liver cells it seems that they are coupled both to the adenylate cyclase-cyclic AMP and the polyphosphoinositide messenger systems. Circulating blood cells might faithfully reflect the insulin receptor state of target organ tissues. Impaired platelet function has an important role in the pathogenesis of vascular and thrombotic complications in diabetes mellitus, and insulin seems to act directly on platelets. A reduction in the number and binding capacity of platelet insulin receptors in diabetic patients (Udvardy et al. 1986) suggested a (patho)physiological role for these receptors. In our studies, insulin (1 × 10-9 - 1 × 10-6 M) did not affect basal platelet cyclic AMP levels, as measured following incorporation of [3H] adenine. Insulin did not prevent PGI2 (25-75 nmol/L) induced cyclic AMP formation in platelets. Insulin did not modify the basal levels of inositol phosphate (IP), IP2 or IP3 in platelets, as measured following incorporation of [3H] inositol. Insulin did not affect formation of IP, IP2 or IP3 by thrombin. No changes in cytosolic free Ca2+ (Quin 2 method) were detected in the presence of insulin. Sodium nitroprusside on the other hand, which is known to mimic several effects of insulin on adipocytes, inhibited IP formation induced by threshold concentrations of thrombin.

On the basis of our results the insulin receptors in human platelets seem to be “non-functional” insofar as their occupancy is not accompanied by the stimulation or inhibition of phospho-inositide breakdown or cyclic AMP formation. Similarly, “silent” muscarinic-cholinergic receptors have recently been reported in human erythrocytes (Sehar et al. 1986).
EFFECT OF EXTRACELLULAR Ca2+ UPON AGONIST-INDUCED Ca2+ TRANSIENTS IN HUMAN PLATELETS: COMPARISON OF QUIN 2 AND FURA 2
Authors: Watts I S , Keery R J , Lumlev P
Published: August 2018

In human platelets loaded with the Ca2+ indicator Quin 2 (Q2), elevated cytosolic Ca2+ ([Ca2+]i) induced by platelet agonists is greatly attenuated in the absence of extracellular Ca2+ ([Ca2+]o), suggesting the majority of [Ca2+]i is derived via transmembrane influx. However, the relatively weak fluorescent properties of Q2 require that high intraplatelet concentrations be used which may influence [Ca2+]i homeostasis and platelet function. This is less of a problem with the more intensely fluorescent Ca2+-indicator Fura 2 (F2). We have compared the rise in [Ca2+]i and aggregation induced by a range of agonists at different [Ca2+]o concentrations, in Q2-and F2-loaded platelets. Platelets in citrated PRP were loaded with the acetoxymethyl esters of Q2 (20 μM) or F2 (4 μM) (30 min at 37°C) and then gel-filtered using a low Ca2+ (≃50 μM) buffer (Ca2+L). Platelet count was 1.5×l08 ml-1. Fluorescence was measured at Ex 339 Em 500 nM in aliquots of platelet suspension at 37°C. Aggregation was determined at 37°C by turbidometric and platelet counting techniques. In Q2- and F2-loaded platelets in 1 mM [Ca2+]o, ADP (0.1 to 10 μM), U46619 (0.01 to 1 μM), PAF (0.001 to 1 μM) and thrombin (0.001 to 0.3 Uml-1) caused concentration-related increases in [Ca2+]i and aggregation. In Q2-loaded platelets, the rise in [Ca2+]i to thrombin, ADP, U46619 and PAF was reduced > 90% in both Ca2+L medium and in the absence of [Ca2+]o (4 mM EGTA). In contrast, in F2-loaded platelets, attenuation of the rise in [Ca2+]i to each agonist in Ca2+L and EGTA was much less than in Q2-loaded platelets (e.g. with U46619 (1 μM) only 20% and 40% reduction in Ca2+L and EGTA respectively). With Q2, aggregation was abolished in the presence of EGTA and markedly inhibited in the Ca2+L medium. In contrast with F2 whilst the presence of EGTA abolished aggregation, no inhibition was seen in the Ca2+L medium. In conclusion, agonist stimulation of platelets is associated with an influx of Ca2+, which is greater in Q2- than F2-loaded platelets. In low [Ca2+]o Q2 renders platelets more reliant on Ca2+ influx for aggregation to occur than does F2. Caution should therefore be exercised when correlating [Ca2+]i with aggregation in Q2-loaded platelets.
CHARACTERIZATION OF DISORDERS OF PLATELET-VESSEL WALL INTERACTION IN AN AGGREGOMETER INCORPORATING BLOOD FLOW PAST AN ENDOTHELIAL CELL MONOLAYER
Authors: Grabowski E F , McKenny K
Published: August 2018

Epi-fluorescence videomicroscopy permits real-time imaging of platelet (plt) adhesion-aggregation to a defined microinjury site of an endothelial cell monolayer (ECM) exposed to flowing blood. The fluorescent label is the TAB murine monoclonal antibody (courtesy of Dr. R.P. McEver) directed against human pit cp HB, together with a fluorescein-conjugated goat F(ab')2 against murine immunoglobulin. The combination assures specificity for pit membranes, yet leaves pit function intact. Bovine aortic ECM, grown on rectangular cover glasses, comprise one wall of a flow chamber mounted on a vertical microscope stage. A 6-0 sterile suture, drawn across the ECM in a direction transverse to flow, creates microinjuries of width 70 ± 15 (mean ± SD). Pit deposition is virtually absent upon intact and confluent regions of the ECM. On microinjury sites and at a shear rate of 270 sec-1, however, computer-enhanced images show pit adherence, aggregation, and embolization. Pretreatment of the ECM with 1.0 mMFC lysine acetyl salicylate, further, leads to a three-fold increase in aggregate length. ECM products inhibitable by aspirin, therefore, modulate adhesion-aggregation in disease and normal states under physiologic flow conditions. The Table shows that nercent coverage of the injury area, and mean aggregate length readily discriminate normal, post-aspirin, and von Willebrand's (vWD's) bloods. Aggregate length is reduced in vWD's blood to a greater degree (p<0.01) than by oral aspirin, while the latter is associated with a paradoxic increase (p<0.01) in single plt adhesion.
COMPUTERIZED PLATELET AGGREGOMETRY
Authors: Szabados T , Hermán F , Kollányi G , Hadházy P , Magyar K
Published: August 2018

One of the commonly used in vitro tests for assessing platelet function is a photodensitometric assay first established by Born. However the collection and analysis of aggregation data are tedious and time consuming. The single- and dual-channel aggregometers have limited utility for the analysis of large numbers of plasma samples in clinical laboratories or in studies on the mode of action of drugs.

We now report on a multichannel platelet aggregome-ter system consisting of an IBM XT personal computer and three microprocessor-controlled 4 channel aggregometers. The system collects, displays and analyzes 12 different aggregation curves simultaneously, and - like other computerized systems - (1) significantly increa ses the efficiency and ease in performing the experiment, analyzing and presenting the data; (2) provides systematic storage and rapid retrieval of the data;(3) saves an enormous amount of time; (4) because of multichannel capability eliminates the effects of time-related changes in PRP on the dose-response curves.

However our system has some advantages over the computerized aggregometer systems used so far: (1) since the aggregometers contain built in microprocessors they can be utilized to measure and analyse platelet aggregation without being coupled to a computer; (2) a special program helps to check the validity of the calculated parameters under visual control; (3) the individual points of the dose-response curves can be checked at any time during the experiment.