bluuebunny/crossref_metadata_2025 huggingface dataset:
BOVINE PULMONARY ARTERY ENDOTHELIAL CELLS (BPAECs) STIMULATE TISSUE TYPE PLASMINOGEN ACTIVATOR (tPA) RELEASE BY HUMAN MELANOMA CELL LINES (HMCLs)
Authors: Wojta J , Vetterlein M , Eoover R N
Published: August 2018

It is known that treatment of endothelial cells (ECs) with extracts of malignant cells results in an increased level of tPA release by such ECs. On the other hand, no data are available concerning an influence of ECs on the fibrinolytic activity (PA) of malignant cells. Therefore it was the aim of this investigation to study whether the tPA production of malignant cells is influenced by ECs. Eighteen different HMCLs were screened for their ability to produce tPA using a combined assay system for tPA activity and tPA antigen employing monoclonal anti tPA antibodies. All 18 HMCLs produced tPA antigen ranging from 0.2 to 15.5ng/105 cells/24 hours. Twelve of these cells produced also tPA activity from 0.2 to 5.0 IU/105 cells/24 hours. To determine the influence of ECs on the PA of HMCLs, BPAECs were selected because their PA consists mainly of urokinase type plasminogen activator (uPA). All 18 HMCLs were cultivated on monolayers of BPAECs and as a control on plastic. After 24 hours the average tPA antigen level in the supernatant for the HMCLs grown on monolayers of BPAECs was 4.2ng/105 cells as compared to 2.9ng/105 cells of the control. However, the tPA activity average level was < 0.1 IU/105 cells for the HMCLs grown on BPAECs and 0.9 IU/105 cells for the HMCLs grown on plastic. To further investigate these effects two HMCLs were grown on plastic and incubated with conditioned media (CM) of BPAECs and compared to non conditioned medium (NCM). After 24 hours the levels of tPA antigen in the supernatant were 194 and 28.5ng/l05 cells after incubation with CM and compared to 14.0 and 14.9ng/105 cells for the control. In contrast to the first experiment the tPA activity level was also increased after incubation with CM. The respective values were 9.5 and 11.6 IU/105 cells and 3.8 and 4.8 IU/105 cells for the control. Our data suggest that BPAECs release a "factor" which is stimulating both the release of tPA activity and tPA antigen. This "factor" remains to be further characterized. On the other hand our results also suggest that BPAECs might release a plasminogen activator inhibitor (PAI) in its active form inhibiting the tPA simultanously released by cocultivated HMCLs.
FIBRINOLYTIC EFFECT OF ONE-CHAIN TTSSUE-TYPE PLASMINOGEN ACTIVATOR
Authors: Johannessen M , Nielsen F E , Pingel K , Petersen L C
Published: August 2018

The fibrinolytic properties of authentic one- and two-chain recombinant tPA were compared to those of a plasmin resistant one-chain tPA analogue, tPA-Gly275, which is point mutated in Arg275 of the activation site. The proteins were characterised by reversed phase HPLC, reduced SDS-PAGE, and their concentrations determined by the BCA (modified Lowry) method. When equivalent conc. of these enzymes were tested for fibrinolytic activity by means of clot lysis and fibrin plate lysis methods, the values found for two-chain tPA were consistently 50% higher than one-chain tPA forms. The time course for plasmin catalysed one-chain tPA cleavage during fibrin clot lysis was determined by means of 125I-tPA. The cleavage is not instantaneous, and one-chain tPA may account for a considerable fraction of the total amount of plasmin formed. This is confirmed by similar experiments with 125I-tPA-Gly275, which is essentially intact one-chain tPA at the time of fibrin clot lysis. The effect of tPA activation site cleavage was also studied using plasmin coupled to sepharose beads. Plasmin sepharose was removed at different time intervals, and tPA was tested for amidolytic activity with > Glu-Gly-Arg-pNA and fibrinolytic activity as measured by fibrin clot lysis time as well as by fibrin plate methods. The results indicate that in the presence of fibrin, plasminogen can be activated by one-chain tPA at a considerable rate. On the other hand, the fact that the fibrinolytic activity measured by conventional assays is lower with one-chain tPA than with two-chain tPA should be considered when of these methods are used for standardization.
COMPARISON OF SINGLE-CHAIN AND TWO-CHAIN RECOMBINANT TISSUE-PLAS-MINOGEN ACTIVATOR (t-PA) IN VITRO. TURNOVER OF SINGLE-CHAIN t-PA IN ANIMALS AND MAN
Authors: Nguyen G , Bara L , Samama M , Conard J , Van Dreden P , Desnoyers P , Bendetowicz A V
Published: August 2018

Two preparations of recombinant t-PA obtained from mammalian cells (Boehringer Ingelheim) containing mainly single-chain forms t-PA (sct-PA > 95%) or two-chain forms t-PA (tct-PA > 70%) were compared in vitro for their thrombolytic and fibrinogeno-lytic properties.

Thrombolytic activities studied on human hanging clots were similar for both preparations, at plasma concentrations ranging from 62.5 to 1,000 IU/ml. Fibrinogenolysis appeared for both preparations at concentrations higher than 500 IU/ml, after a 90 min incubation at 37°C. For higher concentrations, fibrinogenolysis was dose-dependent, and- there was a trend with sct-PA to provoke less fibrinogenolysis.

The turnover of sct-PA was studied in rabbits and cats. After an intravenous bolus injection of 0.2 mg/kg, t-PA activity and antigen showed a double decay disappearance curve. Initial and secondary phases had similar t.1/2 for the activity and the antigen, as well as in both species, which were respectively estimated about 2 min and 20 min.

Although the biological and immunological clearance rates were not significantly different, sct-PA antigen values were much higher than the activity.

Sct-PA clearance rate was also measured in patients with acute myocardial infarction, treated with intravenous infusion of sct-PA, 100 mg over 3 hours, of which 60 mg during the first hour (n=4). t-PA antigen was regularly measured within 20 min after the end of the first hour infusion.

A biphasic disappearance curve was observed, the first phase was extremely rapid with a t.1/2 of 3.2 ± 1.2 min, and the secondary phase had a t.1/2 of 10.4 ± 2.6 min.
EFFECT OF THROMBIN-CLEAVAGE OF PRO-UROKINASE ON ITS AFFINITY TO FIBRIN
Authors: Eguchi Y , Sakata Y , Matsuda M , Kondo K
Published: August 2018

Pro-urokinase (PUK) has been reported to bind to fibrin-Sepharose specifically but not to fibrin clot. As thrombin (TH) clotted fibrinogen and cleaved the Arg 156-Phe 157 peptide bond of PUK simultaneously, we tested effect of this cleavage on the binding of PUK to fibrin by using high molecular weight urokinase (HMUK), PUK purified from culture media of human kidney cell (nPUK) and two kinds of recombinant PUK (rPUK) expressed in genetically transformed E. Coli. One rPUK designated as P0 lacks the carbohydrate side chain present in nPUK but has the same amino acids sequence as nPUK does. The other rPUK designated as P3 is a mutant whose Phe 157 was replaced by Asp so as to be resistant against the proteolytic cleavage by TH. The rate of conversion of single-chain 125I labeled PUK (125I PUK) to two-chain form by TH or plasmin (PM) was determined by both amidolytic activity toward S-2444 and autoradiography of polyacrylamide gel under reducing conditions. Although 100 U/ml TH did not cleave 125I P3 in 2h at 37°C, 10 U/ml TH completely cleaved H00 U/ml 125I nPUK and PO instantaneously. 125I PUK, which had been totally or partially cleaved by either TH or PM, was mixed with fibrinogen in the presence of aprotinin. Then the mixture was clotted with 2.5 mM Ca2+ and 0.5 U/ml TH which cleaved PUK only a little for 10 min. Clots were obtained by clot-syneresis and washed with Tris-buffer containing albumin, aprotinin and hirudin. The bound PUK was calculated by measuring the radioactivity of the clot and the supernatant. Although the binding of nPUK and PO to fibrin decreased from 12% to 1% as cleavage by TH or PM proceeded, that of P3 to fibrin decreased only when two-chain conversion was processed by PM. HMUK did not bind to fibrin. These results strongly suggest that single-chain PUK binds to fibrin and imply that P3, which is TH-resistant, may be useful as an activator in the presence of TH surrounding the clot.
SYNERGY OF TRANEXAMIC ACID AND FIBRIN IN THE ENHANCEMENT OF THE ACTIVATION OF GLU-PLASMINOGEN BY UROKINASE
Authors: Sugawara Y , Takada Y , Takada A
Published: August 2018

The activation rate of Glu-plasminogen (Glu-plg) by urokinase (UK) was enhanced in the presence of either tranexamic acid or fibrin with increase in catalytic rate constant (kcat) without change in Km. The values of kcat increased almost linearly up to 0.5 mM of tranexamic acid concentration and reached a plateau. On the other hand, the presence of increasing concentrations of fibrin resulted in almost hyperbolic increase in kcat values, reaching the plateau level at 0.04 μM. The values of kcat increased three fold at 0.5 mM of tranexamic acid and at 0.04 μM of fibrin. Fibrinogen was less effective and kcat increased two fold in the presence of 1 μM of fibrinogen. Similar kinetic parameters suggest that basic mechanisms underlying the enhanced activation of Glu-plg by UK may be similar between in the presence of tranexamic acid and fibrin. Possibly conformational changes of Glu-plg upon interaction of its lysine binding sites (LBS)< with tranexamic acid or fibrin may be reasons for the enhanced activation. The addition of fibrin to 1 mM tranexamic acid resulted in further increase in kcat of the UK activation of Glu-plg. On the other hand, the addition of tranexamic acid to 0.1 μM of fibrin further increased kcat of the UK activation of Glu-plg. Thus synergy was observed in the activation of Glu-plg by UK between tranexamic acid and fibrin. Fibrin binding site on kringle 5 of Glu-plg may be involved in further increase in the activation rate of Glu-plg by UK in the presence of both fibrin and tranexamic acid in comparison to that in the presence of tranexamic acid alone. Possibly, Glu-plg bound with both tranexamic acid and fibrin (at kringle 5) may be most effectively activated by UK. From the concentrations of fibrin to show 50 % changes of kcat, it is also suggested that two molecules of Glu-plg should bind to one molecule of fibrin monomer.
Kinetics of piasminogen-activation. Effects of ligands binding to the AH-site of plasminogen
Authors: Christensen Ulla
Published: August 2018

Detailed kinetic studies of the urokinase catalysed conversion of Lys-77- and Val-440-plasminogens in the presence and absence of ligands binding to the AH-site of the plasminogens shows that the effects of such ligand-binding correspond with a model of the activation reaction in which the effective Km and kc decreases, but kc/Km increases when the ligands bind. Apparently plasminogen with a free AH-site is a less specific substrate for urokinase, than is plasminogen with an AH-site-bound ligand.

The AH-site is a weak lysine binding site of plasminogen located in the mini plasminogen part (Val-440-Asn-790) of plasminogen and is suggested to participate in the binding of the plasminogens to undegraded fibrin.
DETERMINATION OF FREE CIRCULATING t PA AND MAXIMAL t PA CONTENT IN NORMAL HUMAN PLASMA USING A SPECTROPHOTO-METRIC METHOD
Authors: Boutière B , Arnoux D , Sampol J , Masson C , Hamon F , Anglès-Cano E
Published: August 2018

Recent studies indicate that tPA released by endothelial cells circulates in plasma as a complex with its inhibitor (PAI). Quantitative data have been difficult to obtain however, due to the interference of plasmin inhibitors and other activators on liquid-phase tPA assays. In the present study an activity test (SOFIA-tPA ; Anal. Biochem. 133:201, 1986) on solid-phase fibrin has been used to determine the physiological forms of tPA in normal plasma. In this assay an affinity separation step allows the selective binding of tPA to fibrin and the elimination of plasmin inhibitors and other activators (pro UK, UK). Fibrin-boypd tPA is subsequentely detected by adding plasminogen and a chromogenic substrate selective for plasmin. Under these conditions the reaction rate depends on the presence of tPA, tPA-PAI complexes and PAI present in plasma. Plasma from 38 normal human volunteers obtained before and after venous occlusion was tested. Free tPA was determined in undiluted plasma while the fibrinolytic potential (maximal tPA activity) of plasma was detected following dissociation of tPA-PAI complexes by dilution and acidification of plasma, i.e. euglobulin fractionation. A 1:2 to 1:5 dilution of euglobulins was necessary to obtain a maximal dissociation of precipitated complexes. A similar dilution induced dissociation of tPA-PAI complexes was obtained with plasma at pH 6.8. The amount of free detectable tPA increased as a function of the dilution of plasma until a plateau value (usually at dilution 1:20), which was similar to that observed with the euglobulins. Our results (see Table) indicate that most of the tPA in human plasma circulates as tPA-PAI complexes and that free tPA can be detected only in minute amounts. Plasma instead of euglobulins can be accurately used to detect both free (undiluted plasma) and total tPA (plasma diluted 1:20).
FIBRINOLYTIC RESPONSE AFTER VENOUS OCCLUSION AND DDAVP IN PATIENTS WITH DEEP VENOUS THROMBOSIS
Authors: Kozak M , Keber D
Published: August 2018

After prolonged stimulation venous endothelium becomes refractory to otherwise efficacious stimuli for tissue plasminogen activator (t-PA) release. When the stimulus is removed, the restitution of the response is observed. The distention of the veins distally from the occlusion site in deep venous thrombosis (DVT) could represent such a chronic stimulus. We studied t-PA release in patients with DVT during 20-min venous occlusion (VO) and DDAVP infusion (0,4 ug/kg b.w. in 10 min). t-PA release was estimated as the difference in euglobulin clot lysis time (ECLT in U) and fibrin plates (FP in mm2) before and after VO (fibrinolytic potential). Two groups of patients were studied: 15 recumbent patients with oneside iliofemoral DVT, and 15 patients with oneside postthrombotic syndrome.

In both groups the response of legs was lower than that of arms. The comparison of the healthy and the affected leg in postthrombotic group showed no difference in t-PA release after VO. A higher release of t-PA was seen in the acute DVT group in all three tested limbs, explainable by a restitution of fibrinolytic potential due to the reduction of hydrostatic stimulus. However, the response was slightly lower in the diseased leg. In 5 patients from the acute DVT group DDAVP infusion induced higher t-PA release after VO in both legs compared to VO before DDAVP. We can conclude that hydrostatic pressure in upright position is such a strong stimulus that an additional decrease of t-PA release due to chronic venous stasis cannot be expressed. In recumbent patients DVT hinders the restitution of the response to V0 in the diseased leg. DDAVP seems to act independently of hydrostatic pressure and venous stasis.
PURIFICATION AND CHARACTERIZATION OF PAI-1 FROM HUMAN ENDOTHELIAL CELLS
Authors: Booth N A , MacGregor I R , Hunter N R , Bennett B
Published: August 2018

Platelet α-granules and endothelial cells contain an inhibitor of plasminogen activator, which inhibits both t-PA and u-PA. The inhibitor (PAI-1) is detectable after SDS-PAGE and zymography on fibrin/plasmin-ogen/u-PA detector gels. We have purified endothelial PAI-1 by a simple two-step procedure. Serum-free conditioned medium from human umbilical vein endothelial cells, grown in microcarrier culture, was fractionated on Sephadex CM-50, a cation exchanger, followed by gel filtration on Sephacryl S-200. Aprotinin was included throughout the procedure to maintain the activity of the inhibitor. The PAI-1 was purified 2000-fold with a recovery of about 7%. The purified protein had a specific activity of 8500 U/mg protein and the activity could be stimulated 14-fold by 4M guanidine. The purified PAI-1, of M 48000, was a single-chain glycoprotein.The ptoduct wasapparently homogeneous on a silver-stained SDS-polyacrylamide gel, the protein band co-migrating with PAI activity. Further, a rabbit antiserum raised against the purified PAI-1 revealed only a single band on immunoblots of material from each stage of the purification. The immunoglobulin fraction ofthe antiserum, incorporated into the detector gel for zymographic analysis, neutralised the inhibitor from plasma, platelets and endothelial cells, confirming their identity. Preincubation of PAI-1 from these sources with the immunoglobulin prevented formation of a complex with t-PA or u-PA. This purification procedure, in which no denaturants are employed, provides a homogeneous preparation of PAI-1 that is useful for studies on the stimulatory effects of denaturants. The antiserum raised has allowed the development of a sensitive ELISA, specific for PAI-1.
EFFECT OF DEXTRAN ON PLASMINOGEN ACTIVATOR INHIBITOR (PAI)
Authors: Saldeen T , Moalli R , Hasan F M , Carvalho A , Eriksson M
Published: August 2018

Adult respiratory distress syndrome (ARDS) is often associated with impaired fibrinolysis and appearance of microemboli in the lung. Increased production of PAI, associated with acute lung injury, may contribute to this process. In patients at risk of developing microembolic complications, aeteinistration of dextran reduced the incidence of ARDS. Accordingly, we studied the effect of dextran adninistration on plasma PAI levels in a rabbit model of endotoxin-induced lung injury and in patients. E coli endotoxin (500 μg/kg bw, infused over 5 min) was administrered to three groups of anesthetized, ventilated rabbits; 10 rabbits were pretreated and maintained with a 10% dextran-40 infusion (20 ml/kg bw), 6 received a 3% albunin solution (20 ml/kg bw) and 8 control rabbits received saline. Plasma PAI levels were measured by chrcmogenic assay. By 180 minutes after endotoxin infusion, PAI levels increased from 28 ± 6 to 155 ± 7 U/ml in the saline group, to 152 ± 10 U/ml in the albumin group and to 85 ± 8 U/ml in the dextran group.

24 surgical patients were given 500 ml Dextran 70 (Macrodex) and PAI and t-PA were determined before, during and after dextran. PAI levels decreased 6 ± 8 U/ml during dextran infusion and 11 ±11 U/ml (26 per cent) after dextran, and was significantly decreased after correction for hemodilution. t-PA antigen levels were not changed due to dextran. Dextran thus has an effect on PAI levels which is not due to release of t-PA. Dextran by decreasing PAI levels may have unique salutary effects on fibrinolysis which may be important in the prevention of thrombotic disorders.
EFFECT OF TRANSFORMING GROWTH FACTORβ(TGFβ) ON THE FIBRINOLYTIC SYSTEM OF CULTURED BOVINE AORTIC ENDOTHELIAL CELLS (BAEs)
Authors: Mimuro Jun , Loskutoff David J
Published: August 2018

TGFβ influences cell growth and differentiation in a variety of normal and transformed cells and was recently shown to have growth inhibitory activity toward cultured endothelium. The fibrinolytic activity of endothelial cells changes dramatically with their growth state. Experiments were thus performed to determine the effect of TGFβ on the fibrinolytic system of these cells. Confluent BAEs were incubated with increasing concentrations of purified human or porcine TGFβ for various times in the presence or absence of serum. Conditioned medium (CM) and extracellular matrix (ECM) were prepared and assayed for tissue-type plasminogen activator (tPA), urokinase-type PA (uPA) and PA inhibitor-1 (PAI-1) by immunological methods and by fibrin and reverse fibrin autography. TGFβ induced a dose and time dependent increase in PAI-1 antigen and activity. The half-maximal effect was observed at 0.5 ng/ml while the maximal effect was at 2-5 ng/ml and resulted in 100-fold increase of PAI-1 in CM and at least a 20-fold increase in ECM. 35S-methionine labeling experiments indicated that these effects (1) could be detected in CM within 3-4 hrs, (2) resulted from an increased rate of synthesis of PAI-1, (3) were also observed following transient exposure (1-2 h) of the cells to TGFβ, and (4) were relatively specific for PAI-1. TGFβ treated cells had normal amounts of tPA activity but greatly reduced levels of uPA activity. Human serum had a synergistic effect on PAI-1 production by these cells, but neither epidermal growth factor nor platelet derived growth factor altered their response to TGFβ. TGFβ also stimulated PAI-1 production by cultured human umbilical vein endothelial cells. The release of TGFβ by platelets may thus suppress the fibrinolytic system of the vascular wall.
PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO PROTEASE NEXIN
Authors: Steven I , Wagner L , van Nostrand W E , Cunningham D D
Published: August 2018

Protease nexin I (PN-1) is a cell-secreted protease inhibitor which modulates the activity of certain serine proteases in the extracellular environment. To probe PN-1 functions, we prepared a panel of monoclonal antibodies (mAbs) against it and studied their effects on a variety of PN-1 activities. Three highly potent inhibitors of PN-1 activity have been characterized. MAbs pl-6 and pl-9 stoichiometri-cally block PN-l-mediated anti-thrombin and anti-urokinase activity. These mAbs block PN-l-mediated anti-trypsin activity less effectively. MAb pl-18 stoichiometrically blocks all PN-1 antiprotease activities tested including anti-trypsin activity and therefore appears to bind at the reactive center of PN-1. Heparin, which greatly accelerates PN-1 anti-thrombin activity, does not compete with the binding of these blocking mAbs. None of these blocking mAbs could bind to thrombin-PN-1 complexes. A mAb (pl-1) which did not block PN-1 activity was capable of binding to these complexes.

MAbs pl-9 and pl-18 were used to immunopurify two forms of PN-1 which have different affinities for heparin-Sepharose. These have been referred to as the low heparin affinity and high heparin affinity forms of PN-1 (Scott et al., J. Biol. Chem. 260,7029-7034 [1985]). MAb pl-18 binds only to the high heparin affinity form while mAb pl-9 binds to both forms. Preliminary characterization of this low heparin affinity form reveals no major differences between it and the well characterized high heparin affinity form.
LEVELS OF PLASMINOGEN ACTIVATOR INHIBITOR IN PATIENTS WITH ANGINA PECTORIS
Authors: Koringer C , Jäger R , Huber K , lechner K
Published: August 2018

Several groups have shown that fibrinolytic capacity is impaired in survivors of myocardial infarction, due to increased levels of the fast-acting plasminogen activator inhibitor (PAI). In order to study the behaviour of PAI in patients with coronary heart disease, 180 patients with angina pectoris were investigated. They were 148 males and 32 females, ages ranging from 29 to 70 years (52.8 ± 8.2, mean ± S.D.). A sex- and age- matched normal population served as a control (n=105, age-range 30 to 69 years, 52.4 ± 7.9). PAI was determined by a functional titration assay, and its activity expressed as arbitrary units (AU). PAI levels were significantly (p <0.005) higher in patients with angina (24.3 ± 10.3 AU/ml, range 10.1 to 112.0 AU/ml) than in normals (20.4 ± 4.6 AU/ml, range 10.5 to 31.6 AU/ml). PAI levels were unrelated to sex or age, in both the patient and the control groups. As expected, plasma triglyceride levels were correlated to PAI in patients (r=0.19, p<0.01) and in normals (r=0.20, p<0.05). Patients with a history of previous myocardial infarction (n=114) had similar PAI levels as patients without infarction (24.2 ± 11.1 AU/ml as compared to 24.4 ± 9.6 AU/ml). It is concluded that PAI levels are elevated in patients with coronary heart disease, whether myocardial infarction has taken place or not.
OCCURRENCE OF PAI-2 IN MEN AND NON-PREGNANT WOMEN
Authors: Lecander I , Åstedt B
Published: August 2018

PAI-2 is a specific plasminogen activator inhibitor most likely produced in the trophoblasts of placenta but presumably also in certain macrophages. It occurs in two molecular froms of 48 and 60 kDa. From placenta homogenates mainly the LMW-form can be isolated. During pregnancy mainly the HMW-form increases in the blood and disappears after delivery.

We examined blood samples from 120 male blood donors (females excluded to avoid unknown pregnancies) and 20 staff members, 4 males and 16 females for the presence of PAI-2.

PAI-2 antigen levels were measured with a sandwich ELISA using a polyclonal and a monoclonal antibody. The concentration was given in per cent of that in pooled term plasma.

PAI-2 antigen was detected in 5 of the blood donors 9, 12, 22, 31 and 60 %. In one male staff member the antigen concentration was 9 % and in one female 72 %. Repeated analyzes of this last member for six months resulted in values between 30-90 %. Immunoblotting using a monoclonal antibody against PAI-2 showed a HMW-band of about 80 kDa.
PLASMINOGEN ACTIVATOR INHIBITORS OF TYPE 1 (PAI-1) AND TYPE 2 (PAI-2) ARE PRESENT IN ASCITIC FLUID FROM PATIENTS WITH OVARIAN CARCINOMA
Authors: Lecander I , Martinsson G , Nielsen L S , Andreasen P A , Danø K , Åstedt B
Published: August 2018

The fast acting specific plasminogen activator inhibitor PAI-1 produced in endothelial cells and present in plasma and thrombocytes is known also to be released from certain tumour cell lines. The specific plasminogen activator inhibitor PAI-2 of placental type and present in pregnancy plasma is also released from a histocytoma cell line.

We examined ascitic fluid for the presence of PAI-1 and PAI-2 in five patients with advanced ovarian carcinoma. The antigen levels of PAI-1 and PAI-2 were assayed with sandwich ELISAs using a combination of monoclonal and polyclonal antibodies and the activity of PAI-1 was in addition measured by an activity assay (BioPool). The antigen level of PAI-1 ranged between 110 and 800 ug/1 (mean 315 μg/1) and the activity between 5 and 16 U/ml (mean 12 U/ml). Antigen levels of PAI-2 ranged from 10 to 62 ug/1 (mean 30 μg/ml), i.e. approximately 35 (12-72) % of the concentration of PAI-2 in term plasma. In blood samples obtained from three of the patients during surgery from veins draining the tumors, the concentration of PAI-2 was 0, 7 and 12 μg/1, respectively. PAI-2 could not be detected in the peripheral blood.
STUDIES OF A HIGH AFFINITY, STABLE PLATELET-THROMBIN COMPLEX FORMED DURING PLATELET ACTIVATION. A POSSIBLE MECHANISM FOR TRANSMEMBRANE SIGNAL GENERATION
Authors: Knupp C L
Published: August 2018

The precise mechanism of signal transmission from the platelet surface to the interior required for thrombin-induced platelet activation is not known. A stable complex between Iodo-[125l]-thrombin and platelets has been noted previously on SDS polyacrylamide gels. This 77,000 dalton complex was thought to represent the high affinity binding found in binding studies. The complex is inhibited by excess native thrombin but not by excess active site-modified thrombin. Thus, it displays the characteristics of the crucial thrombin-platelet interaction needed for platelet activation. My studies using similar methods for analysis were performed to determine if this interaction might have a role in signal generation for thrombin-induced platelet activation. Thrombin concentration studies with 3 minute incubations demonstrated that formation of the complex occurred only at thrombin doses which result in platelet activation (above 0.1 U/ml). The amount of the complex increased as the thrombin dose increased. Time course studies with thrombin 1 U/ml revealed that the reaction was rapid and was present on platelets after only 5 seconds of incubation. The complex was noted in supernatants but not until 30 seconds. This interaction was not present on platelets chilled to 4°C and was markedly diminished on platelets exposed to the metabolic inhibitors, antimycin A and 2-deoxyglu-cose. The specific thrombin inhibitors, hirudin and dansylargin-ine N-(3-ethyl-l,5-pentanediyl) amide, prevented the complex formation. Addition of either inhibitor after formation of the complex reversed its formation up to 1 minute after the addition of thrombin but not beyond this time. Treatment with trypsin but not chymotrypsin removed this complex from the platelet. It is concluded that this reaction is not simply a binding event as it requires functional platelets and is only formed at thrombin doses which activate platelets. It is an early, specific, surface reaction which requires thrombin binding and active-site domains for its formation. Formation of this complex is consistent with observation of "receptor processing" noted with specific thrombin binding. Therefore, this reaction could have an important function in the signal processing mechanism for thrombin-induced platelet activation.
THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION
Authors: Poll C T , Westwick J
Published: August 2018

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological.
We thank the British Heart Foundation and Ciba-Geigy USA for financial support.
NONENZYMATIC GLYCATI0N OF HUMAN BLOOD PLATELET PROTEINS
Authors: Cohen I , Burk D , Fullertone R J , Veis A , Green D
Published: August 2018

Atherothrombotic events are common in diabetics, and enhanced platelet function is thought to be partly responsible. Since excessive glycation occurs in many proteins of diabetic subjects, we decided to evaluate glycation of diabetic platelet proteins, and especially those proteins known to be involved in platelet aggregation. We believe this is the first report of nonenzy-matic glycation of platelet proteins from patients with poorly-controlled diabetes mellitus. Overall glycation was assessed by the extent of incorporation of [3H]BH4 into fructosyl lysine separated from whole platelet proteins following protein hydrolysis. We studied 12 diabetic patients, all of whom had severe atherothrombotic disease, and 13 normal controls. Fructosyl lysine eluted in the first major radioactive peak and represented 5.7 ± 1.0 S.D. of the total radioactivity in the normal whole platelet samples. Five of the 12 diabetics showed increased radioactive labeling of fructosyl lysine, without a clear pattern of correlation with the levels of glycation of hemoglobin and albumin (r = 0.33 and 0.17, respectively for hemoglobin and albumin). The pattern of glycation of the various platelet proteins in whole platelets, as determined by the incorporation of [3H]BH4 in proteins electrophoretically separated, does not clearly show a selectivity, although myosin and glycoproteins IIb and IIIa have relatively increased levels of [3H]BH4 incorporation. Artificially glycated platelet membranes exhibit glycation mainly in proteins corresponding to the electrophoretic mobility of myosin, glycoproteins IIb and IIIa and actin.

Whether this glycation results in enhanced platelet function and specific loss of these platelets in vascular thrombi is still unclear. Trapping of highly glycated platelets in thrombi may be responsible for the poor correlation between the extent of platelet glycation and the elevated levels of glycated hemoglobin and albumin. (Supported in part by Grant AM13921-26 to A.V.)
STUDIES ON PHOSPHATASES IN HUMAN PLATELETS
Authors: Sakon M , Uemura Y , Suga K , Tsujinaka T , Kambayashi J , Mori T
Published: August 2018

Activation of platelets by various agonists has been ascribed to be associated with phosphorylation and dephosphorylation of specific proteins such as 20K and 47K polypeptide. Although protein kinases such as myosin light chain kinase and C kinase have been extensively studied, little information is currently available on platelet phosphatases, which may play a crucial role in the regulation of stimulus-linked protein phosphorylation. Thereby, the present study was conducted to know some characters of platelet phosphatases. Glycerol loaded platelets prepared from human platelet concentrates were subjected to osmotic lysis in 20 mM HEPES-NaOH buffer containing 5 mM EDTA, 0.5 mM dithio-threitol and various protease inhibitors and a soluble fraction was obtained by centrifugation, The activity of phosphatase was assayed at pH 7.35, using paranitrophenylphosphate as a substrate. Leupeptin and EDTA were added to the reaction mixture to avoid proteolytic attack to the enzyme. The neutral phosphatase was partially purified from the soluble fraction by a combination of ammonium sulfate fractionation and column chromatographies. Five distinct peaks with neutral phosphatase activity were obtained by a linear gradient elution ( 0−0.5 M KCl ) in DEAE Sepharose CL-6B of 0−60 % ammonium sulfate precipitate. The phosphatase activity of one peak eluted at 0.2M KCl was maximum at pH below 6, which was considered to be acid phosphatase, and the remaining four peaks' optimal pH was between 7.0−7.5. These four peaks were termed as PH-I (passed through fraction), PH-II (0.1M KCl), PH-III (0.25M KCl) and PH-IV (0.3M KCl). The respective peak was eluted as a single peak on Ultrigel AcA 34 and the molecular weight was estmated as follows; I-55K, II-40K, III-55K, IV-37K. PH-I − II were active in the presnce of EDTA and were not affect ed by divalent cations (Mg++ , Mn++ , Ca++ ) , whereas PH-III was highly dependent upon Mg++. The activity of PH-IV was completely dependent on Mn++. From these observations, the following conclusions were obtained; (1) Human platelets contain four species of neutral phosphatases, in addition to acid phosphatase. (2) Each neutral phosphatase is distincive by molecular weight and requirement of divalent cations.
A MOLECULAR MECHANISM FOR THE DITHI0THREIT0L-MEDIAT5D PLATELET AGGREGATION
Authors: Jamaluddin Moideen P , Sreedevi C , Thomas Ancy , Krishnan Lissy K
Published: August 2018

Biochemical mechanisms of stimulus response coupling is an intricate problem in platelet biochemistry. Recently we obtained evidence that support the view that conformational changes of an (unsaturated fatty acid – and U46619-binding) haemoprotein induced by the binding of arachidonic acid, H2O2 or PGH2 liberated in apparently different platelet compartments in response to different stimuli could constitute a mechanism (L.K. Krishnan … M.P. Jamaluddin, FEBS Lett, in the press). We investigated the effect of dithiothreitol (DTT), a platelet agonist whose mechanism of action is unknown, on the purified haemoprotein. DTT was found by spectral measurements and gelfiltration experiments to bring about a slow time-dependent conformational .change and oligomerization of the protein concomitantly with its oxidation. Oxidised DTT (trans-4,5-dihy-droxy-1,2-dithiane) was found to induce a similar conformational change by binding to the protein (halfsaturation cancn. 2 mM). Oligomerization changed the charge characteristics of the protein, from net positive to net negative, ait pH 7.4. Protein-protein association is associated with large volume increases. Excluded volume effects and changes in charge distribution at the side of protein conformational change could trigger actin polymerization, pseudopod formation and aggregation, modulated by protein phosphorylation and Ca2+ concentration. In conformity with these ideas oxidized DTT near its half-maximal saturation concentration for the protein, was found to aggregate gelfiltered calf platelets. Presumably it functions as a thioanalogue of PGH2. Oxidized glutathione or oxidized 2-mercaptoethanol could also bring about protein conformational change and platelet aggregation.
ROLE OF PLATELETS IN EXPERIMENTAL THROMBOSIS INDUCED BY VENOUS STASIS
Authors: Bernat A , Vallée E , Maffrand J P , Gordon J
Published: August 2018

Venous stasis in the rat, induced by ligature of the vena cava, provokes thrombosis. This venous thrombosis was initially believed to be platelet-independent because severe thrombocytopenia (95 % reduction in platelet count), aspirin and dipyridamole had little effect. However, the model responded to other platelet anti-aggregators, such as Ticlopidine and its analogue PCR 4099, although these compounds had no effect on coagulation, fibrinolysis or leucocyte functions (Thromb. Res. 37, 279-285, 1985). Both these drugs are known to exert their main antiplatelet effect against aggregation induced by ADP.

The aim of the present study was to re-evaluate the role of platelets in this model of venous thrombosis. We have been able to show that :

1) complete thrombocytopenia (99 %), achieved with an antiplatelet anti-serum, dramatically inhibited thrombus formation (by 84 % ; p < 0.01).

2) partial transfusion of platelets (23 %) from control animals to these thrombocytopenic rats re-established the thrombosis.

3) transfusion (under identical conditions) of platelets from rats treated with PCR 4099 had no effect.

4) vena cava ligature in Fawn Hooded rats (deficient in platelet dense granules) induced less thrombosis (64 % of control ; p < 0.05).

We conclude that this venous stasis model is platelet-dependent. Furthermore, because thrombus formation was reduced in normal rats treated with anti-aggregants acting selectively against ADP, and in rats lacking ADP in their platelet dense granules, it appears that ADP plays a major role in this model of thrombosis.
A ROLE OF DIACYLGLYCEROL KINASE IN STIMULUS-SECRET I ON COUPLING OF HUMAN PLATELETS -DISSOCIATION OF SEROTONIN SECRETION FROM Ca2+ MOBILIZATION-
Authors: TOHMATSU T , NAKASHIMA S , HATTORI H , SUGANUMA A , NOZAWA Y
Published: August 2018

Diacylglycerol (DG) kinase catalyzes the reaction: DG + ATP phosphatidic acid + ADP and it is widely distributed in animal tissues. The enzyme seems to play a pivotal role in removing a second messenger, DG, which activates protein kinase C. DG kinase inhibitor, R 59 022 (6-[2-[4-[(4-fluorophenyl)pheny1- methylene] -1-piperidinyl] ethyl] -7-methyl-5H-thiazolo [3,2-a] -pirimidin-5-one) has recently been developed. In order to gain further insight into the role of DG in the secretory response, effects of the DG kinase inhibitor on secretory responses and Ca2+ mobilization were investigated in human platelets.

The addition of the DG kinase inhibitor (10 μM) potentiated thrombin-induced accumulation of [3H]radioactivity of DG in platelets loaded with [3H] arachidonate. Thrombin-induced release of [3H] arachidonic acid and its metabolites was not affected by the inhibitor. The inhibitor did not cause significant secretion of [14C] serotonin by itself. However, the pretreatment with this agent potentiated the level of secretion in thrombin-stimulated platelets. When l-oleoyl-2-acetylglycerol(OAG) was added to [32pjpi-iabeled platelets in the presence of the DG kinase inhibitor, the formation of [32P] l-oleoyl-2-acetylphosphatidic acid was greatly prevented. The pretreatment with the inhibitor also potentiated OAG-induced serotonin secretion. With the view that Ca2+ is thought to be another important second messenger, we investigated the effect of the DG kinase inhibitor on Ca2+ mobilization. Two types of Ca2+ indicators, Quin2 and aequorin were used to measure cytosolic free Ca2+ concentration ([Ca2+]i). The inhibitor alone did not affect [Ca2+]i. Interestingly, thrombin-induced increase in [Ca2+]i was suppressed by the pretreatment with this agent both in the Quin2-loaded and aequorin-loaded platelets.

These results indicate that diacylglycerol kinase may operate as an attenuator in the signal transduction system involving protein kinase C and that Ca2+ mobilization may not be tightly coupled to serotonin secretion.
R59022, A DIACYLGLYCEROL (DG) KINASE INHIBITOR POTENTIATES THROMBIN-INDUCED PLATELET AGGREGATION AND GRANULE RELEASE
Authors: Joseph S K , Krishnamurthi S , Kakkar V V
Published: August 2018

R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emphasizing the importance of DG in promoting granule secretion. In addition to inhibiting DG phosphorylation, R59022 also inhibits the phosphorylation of the DG analogue 1-oleoyl 2-acetylglycerol (OAG) [1]. OAG (63μM) with pre-incubation times of 10-60 sec, significantly potentiated threshold T (0.03U/ml)-induced [l4C]-5HT release, though with longer incubation times, this potentiatory effect was gradually lost eg 0.03U/ml T-l±0.3%, OAG+T (10 sec)- 33±4%, OAG+T (1 min)-11±3%, 0AG+veh.-0%. However, in the presence of R59022 (30μM), OAG retained its potentiatory effect for longer periods eg R59022+0AG+T (1 min)-45+10%, R59022+T-2±l%. With incubation times > 5 min the potentiatory effects of OAG were lost even in the presence of R59022. This is possibly due to the metabolism of OAG by DG lipase. Our results demonstrate that R59022, which has been reported to inhibit DG kinase leading to enhanced PrkC activation, also enhances agonist-induced platelet aggregation and 5HT release. It may therefore be a useful compound in elucidating further the role of DG in terms of both stimulatory and inhibitory effects on platelet activation.

[1]. de Chaffoy de Coucelles, D. et al (1985) J Biol Chem 260, 15762.
PROTEIN KINASE C CONTROLS CA2+ MOBILIZATION IN HUMAN PLATELETS
Authors: Siffert W , Scheid P , Akkerman JW N
Published: August 2018

Platelet stimulation has been shown to result in a rise of cytosolic pH (pHi) as a result of an activation of a Na+/H+ antiport. We have investigated the role of pH in Ca2+ mobilization in human platelets. pHi and free Ca2+, {Ca2+)i, were measured in platelets loaded with the fluorescent indicators BCECF and quin2, respectively. Stimulation of platelets by either thrombin or OAG, an activator of protein kinase C (Pk-C), increased pHi. Pretreatment of platelets with inhibitors of Pk-C, trifluoperazine (TFP) or sphingosine (SPH), blocked the stimulus-induced rise in pHi, suggesting a role of Pk-C in the activation of Na+/H+ exchange. Blocking Na+/H+ exchange by an amiloride analogue or by TFP similarly suppressed the thrombin-induced increase in {Ca2*}i. This effect could be prevented by increasing pHi with the Na+/H+ ionophore monensin or with NH4Cl. The thrombin-induced (0.05 U/ml) rise in {Ca2+}i was more than 3-fold enhanced when the pH was raised from 6.8 to 7.4.

Our results demonstrate that pHi controls Ca2+ mobilization in human platelets and suggest that Pk-C contributes to this control by activating the Na+/H+ exchanger.

Supported by the Deutsche Forschungsgemeinschaft. No Sche 46/5-2.
THROMBIN-INDUCED RELEASE OF INTRA-PLATELET CA2+ STORES IS INHIBITED BY PROSTACYCLIN, BUT GTP- AND IP3-INDUCED RELEASE IS UNAFFECTED
Authors: Crouch Michael F , Nolan Roger D , Lapetina Eduardo G
Published: August 2018

Alpha-thrombin induced the release of internal Ca2+ stores and the influx of Ca2+ in human platelets, as measured by quin-2 fluorescence. This was accompanied by a stimulated formation of inositol phosphates and phosphatidic acid. The Ca2+ responses were inhibited almost totally by pretreatment of cells with prostacyclin (PGI2). Epinephrine was able to restore the influx of Ca2+ from the external medium, but not the alpha-thrombin-induced release of internal Ca2+ stores. This was despite epinephrine restoring phosphatidic acid formation and, at least partially, the generation of inositol trisphosphate (IP3). This suggested that PGI2 was inhibiting the actions of IP3 in inducing release of Ca2+ from the dense tubular system. Since the effects of PGI2 are thought to be mediated by formation of cAMP, we examined whether cAMP could modulate the release of 45Ca2+ induced by IP3 from permeabilized platelets. IP3 induced about a 30% release of cellular 45Ca2+ over a 4 min period. However, neither pretreatment of cells with PGI2 nor the direct application of dibutyryl cAMP had any effect on the IP3-stimulated 45Ca2+ release. GTP, which released about 10% of total cell 45Ca2+, also was not affected by these agents. These results suggest either that permeabilization of platelets dilutes cytoplasmic components which are necessary for cAMP action, or that PGI2 is inhibiting the release of Ca2+ stores induced by thrombin, presumably via IP3, by a mechanism which is separate to the elevation of intracellular cAMP levels.