bluuebunny/crossref_metadata_2025 huggingface dataset:
INCORPORATION OF PLASMINOGEN ACTIVATOR INHIBITOR INTO FIBRIN, AN ALTERNATIVE REGULATORY PATHWAY OF FIBRINOLYSIS
Authors: Murayama H , Bang N U
Published: August 2018

A plasminogen activator inhibitor (PAI-1) Mj, 50 kd is normally found in plasma at low concentrations. Plasma levels increase sharply upon stimulation of endothelial cells with endotoxin or monokines and activated platelets secrete significant quantities of PAI-1. It is possible that high levels of PAI-1 may be achieved at the local sites of intravascular thrombi. Semipurified PAI-1 was therefore prepared from human platelets to study its affinity for fibrin (F). Approximately 50% PAI-1 adsorbed to F monomer immobilized on sepharose and desorbed under conditions of acidic pH and high ionic strength suggesting hydrogen bonding as the mode of interaction. Wells of 96-well microtiter plates were each coated with 50 yg [125I] plasminogen (P)-free fibrinogen and clotted with thrombin in the presence and absence of different concentrations of PAI-1. After extensive washing of the wells, they were incubated with 5 mU of tissue plasminogen activator (t-PA) and 5 mU of P for 6 h. Appropriate calibration curves utilizing different concentrations of t-PA and different concentrations of PAI-1 added to the supernatant rather than to F established that 8-15% of 21-166 mU PAI-1 incorporated into crosslinked (XL) F or noncrosslinked (NXL) F. Incorporated PAI strikingly inhibited fibrinolysis (FL). Percent inhibition of FL of XL or NXLF (Mean±S.D., N=5) plotted in the presence of 166, 83, 42 and 21 mU of PAI were: 83±3.3, 59.5±1.8, 29.7±5.2 and 15.2±6.14 for XLF and 78±5.3, 31±8.1, 14.5±10.5 and 0 Tor NXL F. As demonstrated by radioautography on SDS PAGE PAI-1 incorporated into F readily formed complexes with [125I] urokinase (u-PA). In these experiments, no evidence for crosslinking of PAI-1 into F has been obtained to date. In experiments utilizing agarose immobilized proteins, it was evident that not only F but also fibrinogen binds PAI-1; PAI-1 associated with F as well as fibrinogen is capable of forming complexes with [1251] u-PA.

In contrast, fibronectin, collagen, gelatin and albumin did not bind PAI-1. Thus, PAI-1 in analogy with alpha-2 plasmin inhibitor may modulate physiological fibrinolysis through incorporation into fibrin.
INCREASED PLASMA LEVELS OF PLASMINOGEN ACTIVATOR INHIBITOR (PA-I) IN OBSTRUCTIVE JAUNDICE (OJ)
Authors: Colucci M , Altomareo D , Chettao G , Cavallo L G , Semararo N
Published: August 2018

Microvascular thrombosis is considered an important pathogenetic factor in renal failure associated with OJ. Plasma levels of PA-I, measured by an amidolytic assay in 29 patients with OJ, were significantly increased as compared with 20 control patients (X ± SEM: 26.5 ± 3.5 vs 6.0 ± 1 units/ml; p < 0.001). Fibrin autography of plasma supplemented with tissue plasminogen activator (t-PA,50 IU/ml) revealed that in jaundiced samples most of the added activator migrated with an apparent Mr of 100 Kd, corresponding to t-PA-PA-I complex, whereas in control samples virtually all t-PA migrated as free enzyme. Rabbits made icteric by bile duct ligation showed an early and progressive increase in plasma PA-I activity (3- and 6-fold higher than the preoperative levels on the 3rd and 10th day respectively) indicating that OJ itself may cause a rise in circulating PA-I. Venous stasis (10 min) in jaundiced patients (n=4) was neither associated with an increase in blood fibrinolytic activity nor with a decrease in PA-I activity. t-PA antigen determination showed that activator release was impaired in 3 out of 4 patients. In controls (n=4), venous occlusion induced a rise in both fibrinolytic activity and t-PA antigen and a reduction in PA-I activity. Finally, bile duct recanalization in patients subjected to surgery (n=7) was accompanied by a decrease in plasma PA-I activity which paralleled the decrease in seric bilirubin levels. It is concluded that OJ, independently of the primary disease, may induce a marked increase in circulating PA-I and perhaps an impairment of t-PA release. Defective fibrinolysis may well contribute to microvascular thrombosis and renal failure associated with OJ.
STUDIES ON THE EFFECT OF CILOSTAZOL ON THE PROSTAGLANDINS PRODUCTION DURING ADP-INDUCED PLATELET AGGREGATION WITH ENDOTHELIAL CELLS
Authors: Chijiwa T , Shiragiku T , Kato S , Igawa T , Kimura Y
Published: August 2018

It has been clinically reported that cilostazol has a potent inhibitory effect on platelet aggregation without changing the prostacyclin level. This study was undertaken to elucidate this clinical effect by a technique developed by us in which platelet aggregation could be evaluated in the presence of cultured endothelial cells. Human umbililical cord vein endothelial cells (HUVEC) were coated (cultured for a few days supplemented with 10% fetal claf serum) on the inner surface of glass cuvette, and platelet aggregation was traced by the stimulation of citrated-PRP with 7.5 μM ADP in this cuvette. The 6-keto-prostaglandin F1α (6-k-PGF) and thromboxane B2 (TXB) produced in the supernatant of the stimulated PRP were measured by radioimmunoassay (Amersham). The cyclic adenosine monophosphate (cAMP) level in platelets and HUVEC was measured by radioimmunoassay (YAMASA). Cilostazol showed a potent inhibitory effect on platelet aggregation in the presence of HUVEC by the suppresion of TXB production, but not by the suppression of 6-k-PGF production. Cilostazol stimulated cAMP production in both platelets and HUVEC. On the other hand, aspirin also showed an inhibitory effect on platelet aggregation in the presence of HUVEC, but suppressed production of both TXB and 6-k-PGF.

As a result, the clinical effect of cilostazol was confirmed by the fact that TXA2 production in a platelet/HUVEC coexisting system was specifically suppressed.
A ROLE OF PLATELET MEMBRANE COMPONENTS IN THE INTERACTION OF PLATELET-COLLAGEN-VON WILLEBRAND FACTOR
Authors: Aihara M , Morimoto S , Sawada Y , Kimura A , Chiba Y , Yoshida Y
Published: August 2018

To determine a role of platelet membrane components on the interaction of platelet-collagen-von Willebrand factor(vWF), several experimental approaches were used. The adhesion of human fixed washed platelets(FWP) to collagen was decreased after the treatment with Serratia marcescens protease(100 ug/ml), but the collagen cofactor activity(COo) of vWF that enhances the adhesion of FWP to collagen was still present after the digestion. Although the platelet adhesion in the absence of normal plasma was not changed by the addition of monoclonal antibody(M-ab) against platelet membrane glycoprotein(GP) IIb/lIIa(1 0E5, BS Coller), the adhesion was decreased by 30-50% after the treatment of the platelets with 10-100 ug/ml anti-GPIb(6D1, BS Coller). The adhesion of FWP to collagen was inhibited by lectins;the adhesion was 58-75% in the presence of 100-400 ug/ml L. culinaris lectin or weat germ agglutinin and the adhesion was nil in the presence of 100 ug/ml Ricinus communis agglutinin I or 200 ug/ml concanavalin A. By the crossed aff ino-immunoelectrophoresis, the binding of GP Ilb/lIIa in Triton-solubilized platelet supernatant to the collagen spacer gel was observed. When CHAPSO solubilized platelet was applied to the collagen column and the fractions containing adhesion inhibitor were eluated by 0.3M NaCl, Mr of 240K, 220K, 21 OK, 116K, 61K, 54K, 50K and 45K proteins were identified besides the proteins which correspond to thrombospondin, GPIb, GP lib or Ilia by SDS-PAGE(7.5% gel, silver stain). GOo in normal plasma was not changed by anti-GPIIb/lIIa but was decreased to 32-38%by anti-GPIb. M-ab against vWF, CLB-RAg 35(van Mourik), that inhibits the binding of vWF to platelet by ristocetin decreased COo in normal plasma by 70% and CLB-RAg 201 (van Mourik) that inhibits the binding of vWF to collagen did completely inhibit the COo in normal plasma. In conclusion, our data suggest that (1) GPIb is partly involved in the platelet adhesion to collagen; (2) the binding of vWF to collagen is required for the expression of CCo; (3) CCo of vWF is partly mediated though GPIb; and (4) several platelet membrane protein(s) besides GPIb or GPIIb/lIIa may be also involved in both the adhesion of platelets to collagen and CCo of vWF.
STUDIES ON THE ANTITHROMBOTIC ACTIONS OF A COLLAGEN PEPTIDE IN A DEFINED MODEL OF VENOUS THROMBOSIS
Authors: Huan X , Legrand Y , Soria J , Soria C , Caen J , Fareed J
Published: August 2018

The synthesis and biologic actions of a collagen peptide Lys-Pro-Gly-Glu-Pro-Gly-Pro-Lys (CP) were first reported by Fauvel, et. al. in 1979. This peptide was found to inhibit collagen induced aggregation of platelets and to modulate clot formation and specific collagen induced fibrinogen binding by platelets. CP exhibited antithrombotic actions in a modified venous stasis thrombosis model (rabbit) against defined thrombogenic challenges, (ED50;(IV) 0.5 − 2.5 mg/kg; (SC) 2.5 − 7.5 mg/kg). The antithrombotic actions of this peptide exhibited a short half-life (10 minutes) after IV administration. Preincubation of this peptide with trypsin, carboxypeptidase A, carboxypeptidase B and pancreatic protease complex resulted in a loss of the antithrombotic action. Specific protease inhibitors were found to protect the peptide from this degradation effect. CP did not exert any effect on the routinely performed global coagulant and antiprotease tests at concentrations which produce antithrombotic actions (0.5 − 5 mg/kg). CP was found to synergize the antithrombotic action of heparin in both intravenous and subcutaneous routes. Preliminary studies on the mechanism of the antithrombotic actions of collagen peptide suggest these actions may be independent of effects on platelets. These studies also suggest that an oligopeptide containing arginine and lysine residues, mimicing serine protease susceptible sites, is capable of producing antithrombotic effects in an animal model of venous thrombosis.
PLATELET AND VESSEL WALL REACTIVITY IN HYPERTENSIVE AND N0RM0TENSIVE RHESUS MONKEYS
Authors: Norden C , Heine H , Misselwitz F , Herrmann H-J , Engler E , Martin G
Published: August 2018

Hypertension is an important risk factor in the pathogenesis of arteriosclerosis and its thrombotic complications. Therefore, we investigated platelet and vessel wall reactivity in 7 subhuman primates (7.0 Macaca mulatta). Five monkeys received a psychosocial stress over a long-term period, three developed a stress-induced hypertension. Two unstressed normotensive monkeys served as e control. Platelet turnover in vivo, synthesis of platelet prostanoids, and various platelet function tests in vitro were determined. Vessel wall reactivity was assessed histo-pathologically and by means of H-thymidin-incorpora-tion into vessel segments in vitro. In the hypertensive rhesus monkeys an accelerated platelet turnover, accompanied with a decreased platelet count, and a markedly increased plasma-TXB2-level were found. ADP-induced platelet aggregation was slightly enhanced. Additionally, platelet adhesion to collagen-coated surfaces was investigated. We found the platelet attachment, spreading, and the formation of platelet mural thrombi to be significantly enhanced in the hypertensive animals. Histopathological examination of large arteries revealed signs of an increased intra-vital vasocontraction as well as enlargement of the relative vessel wall cross-section area in the hypertensive rhesus monkeys. Autoradiographical determination of the thymidine-incorporation in vitro allows investigation of cell proliferation in the intima and media of vessels. We found hypertension-related alterations of the media not only in large, but also in small vessels. Additionally, primary cultures of aortic endothelial cells were established and concentration of endothelial cell-specific metabolites was measured in the conditioned media. Our results confirm the existence of hypertension-related changes in platelet and vessel wall reactivity.
REGULATION OF PLATELET cAMP FORMATION BY PROTEIN KINASE C
Authors: Hopple Sara , Bushfield Mark , Murdoch Fiona , MacIntyre D Euan
Published: August 2018

Exogenous synthetic 1,2-diacylglycerols (e.g. 1,2-dioctanoylglycerol, DiC8) and 4β Phorbol esters (e.g. phorbol myristate acetate, PMA) routinely are used to probe the effects of protein Kinase C (PKC) on cellular responsiveness. Such agents act either independently or synergistically with elevated [Ca2+]i to induce platelet activation, but also inhibit agonist-induced inositol lipid metabolism and Ca2+ flux. These findings led to the concept that activated PKC can function as a bi-directional regulator of platelet reactivity. Therefore, DiCg and PMA were utilized to examine the effects of activated PKC on receptor-mediated stimulation and inhibition of adenylate cyclase, as monitored by cAMP accumulation. All studies were performed using intact human platelets in a modified Tyrodes solution, and cAMP was quantified by radioimmunoassay. Pretreatment (2 min.; 37°C) of platelets with PMA (≤ 300 nM) but not DiCg (200 μM) attenuated the elevation of platelet cAMP content evoked by PGD2 300 nM) but not by PGE1 (≤300 nM), PGI2 (≤100 nM) or adenosine (≤ 100 μM).

These effects of PMA were unaffected by ADP scavengers, by Flurbiprofen (10 μM) or by cAMP phosphodiesterase inhibitors (IBMX, 1 mM) but were abolished by the PKC inhibitor Staurosporine (STP, 100 nM). In contrast, DiC8 (200 μM), but not PMA ( ≤ 300 nM), reduced the inhibitory effect of adrenaline (5 μM) on PGE1 (300 nM)-induced cAMP formation. This effect of DiCg was unaltered by STP (100 nM). Selective inhibition of PGD2-induced cAMP formation by PMA most probably can be attributed to PKC catalysed phosphorylation of the DP receptor. Reduction of the inhibitory effect of adrenaline by DiC8 could occur via an action at the α2 adrenoreceptor or Ni. These differential effects of PMA and DiC8 may result from differences in their distribution or efficacy, or to heterogeneity of platelet PKC.
ACTION OF GTPγS [GUANOSINE 5∲-0-(3-THIOPHOSPHATE)] ON SAPONIN-PERMEABILISED PLATELETS: INVOLVEMENT OF 'G' PROTEINS IN PLATELET ACTIVATION
Authors: Authi K S , Evenden B J , Crawford N
Published: August 2018

Certain ligand-receptor interactions at cell surfaces lead to the phospholipase-C (PLC) hydrolysis of phosphatidyl inositol (4.5) bisphosphate (PIP2). The products serve as intracellular second messengers, e.g. inositol (1.4.5) trisphosphate (IP3) releases Ca2+ from intracellular stores and diacylglycerol activates protein kinase-C. From studies using GTP and analogues (e.g. GTPγS) there is evidence of a key role for a guanine nucleotide binding protein(s) as a link between receptors and PIP2 hydrolysis. We report the actions of GTPγS on washed human platelets permeabilised with saponin (12-14 μg/ml) to allow penetration of low MWt polar substances. The responses to GTPγS are dose dependent (range 9-60 μM) and at 60 μM the agent induces shape change, aggregation and the secretion of 50% of previously incorporated [14C]-5HT. No effect of GTPγS is seen with intact cells. Shape change occurs 25-30 sec after GTPγS; aggregation and secretion is complete after 3 min. When GTP was used (up to 135 μM) with similarly permeabilised platelets no responses were initiated. Phosphatidylinositol turnover was monitored using 32P-labelling before permeabilisation. The addition of 90 μM GTPγS resulted in a 143 ± 23% (n=4) increase in 32P-phosphatidic acid (PA) with respect to the basal levels of “saponised control” cells. These findings suggest that GTPγS stimulates PLC activity through a ‘G’ protein interaction. The GDP analogue (GDPβS) produced no activation responses in saponised platelets but inhibited responses induced by GTPγS in a dose dependent manner (0-480 μM, max inhibition 480 μM). At 960 μM, GDPβS totally inhibited aggregation and secretion initiated by low doses of thrombin (0.1 U/ml) and collagen (1 μg/ml). Identical inhibition by GDPβS of thrombin and collagen-induced activation of intact platelets was observed indicating membrane penetration of this analogue. Shape change effects were not inhibited by GDPSS. The inhibitory effects of GDPSS towards thrombin and collagen induced secretion could be progressively overcome at higher doses of thrombin (0.2 U/ml - 2 U/ml) and collagen (5 μg/ml - 60 μg/ml) suggesting that at higher concentrations these agonists may exert effects through 'G' protein-independent mechanisms.
INHIBITION BY NEOMYCIN OF AGONIST-INDUCED POLYPHOSPHOINOSITIDE METABOLISM AND RESPONSES IN HUMAN PLATELETS
Authors: Holmsen Holm , Tysnes Ole-Bjørn , Verhoeven Adrie J M , Steen Vidar M , Moore Lindsey J , Rongved Sissel
Published: August 2018

Signal processing in platelets seems to involve polyphosphoinositide (PPI) metabolism, although direct coupling between PPI metabolism and responses has not been proved. Neomycin binds tightly to PPIs and has been used to probe the involvement of PPI metabolism and responses in platelets. Neomycin(SO4)3 powerfully inhibited ADP- and adrenaline-induced aggregation of platelets in PRP. This was partly due to the sulphate anion; the chloride form was therefore prepared. Platelets were prelabelled in PRP with 32P-Pi. and transferred by gel filtration to a calcium-free Tyrode’s solution (GFP). Increasing concentrations (2-5 mM) of neomycinCl6 caused progressive inhibition of thrombin-induced aggregation, dense granule secretion, acid hydrolase secretion and formation of 32P-phosphatidic acid (PA); the inhibition was immediate, not affected by aspirin and counteracted by increasing thrombin concentrations. Incubation of neomycin (up to 5 mM) with this GFP or with P-Pi. On GFP prepared from unlabelled PRP had no effect on the P content of ATP, phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4,5-bisphosphate (PIP ). Increasing neomycin concentrations caused progressive inhibition of the thrombin-induced init^l (10 sec) decrease, but not of the late (90 sec) incg^ase i-n P-PIP2 while they enhanced the increase in P-PIP. Similar results were obtained ^th collagen and PAF. Both the increase in cytosolic Ca and pH (measured by INDO-I and BCECF, respectively) induced by thrombin were inhibited progressively by increasing concentrations of neomycin. These results are in support for a direct involvement of PPI metabolism in the stimulus-response coupling below the receptor level. However, the failure of neomycin to affect turnover of PIP and PIP2 in nonstimulated platelets suggests that the aminoglycoside does not penetrate the membrane, and only become available to PPI during stimulation.
LACK OF CORRELATION BETWEEN INTRAPLATELET cAMP INCREASE AND SYSTEMIC CIRCULATORY EFFECTS
Authors: Haarmann W , Weisenberger H
Published: August 2018

Compounds inhibiting platelet function by acting on platelet cAMP metabolism usually also have effects on the circulatory system, i.e. they decrease systemic blood pressure (bp) and are positive inotropic. For several compounds selected because of their distinct platelet inhibitory effects, the influence on these parameters in animals and on the cAMP metabolism in human platelets was determined.

Inotropic effects and bp were measured via an indwelling catheter in anestetised cats after i.v. application of the test compounds. The inhibition of platelet PDEs was measured in freeze-thaw homogenates of human platelets using 3H-cAMP as substrate. Intraplatelet cAMP changes were measured by prelabelling the ATP pool with 3H-adenine and isolation of 3H-cAMP. Linear regression analysis of the drug concentrations causing a doubling of intraplatelet cAMP levis and the % difference in bp or the % difference in dp/dt, resp., by i.v. application of 0.3 mg/kg test compound yielded the following results:

cAMP vs % diff. bp : r=0.02, N=18

cAMP vs % diff. dp/dt: r = 0.02 , N = 15

In contrast to a good correlation between intraplatelet cAMP levels and inhibition of platelet function tests, no obvious relationship was seen between cAMP and decrease in bp and positive initropic effects. It is not known whether the lack of correlation could be due to a different drug access to platelets and the bp regulatory system.

A biochemical parameter, i.e. intraplatelet cAMP increase by inhibition of PDEs correlates reasonably well with the inhibition of platelet function tests. This parameter is not useful, however, to predict the effects on the heart and the circulatory system.
COMPARISON BETWEEN IN VITRO AND IN VIVO AGGREGATION OF PLATELETS
Authors: TAPPARELLI C , GFELLER P , SANJAR S , MORLEY J
Published: August 2018

The extensive utilisation of in vitro tests of platelet aggregation presumes that corresponding effects occur in vivo. Platelet aggregometry in vitro and in vivo have been compared using a range of agonists in order to test this assumption.

PRP from citrated peripheral blood of man, rat and guinea-pig was exposed to ADP, adrenaline, serotonin, collagen, thrombin and PAF in a Born aggregometer to define the time course and amplitude of these responses. In rat and guinea-pig, these aggregatory stimuli have also been used to define the time course and amplitude of intrathoracic accumulation of 111 -Indium labelled platelets, using an automated monitoring system (AIMS 8000).

Concordance between these tests was evident for ADP, collagen, thrombin and PAF in both species; but, substantial discrepancy was observed between in vitro and in vivo responses to serotonin and adrenaline, since sustained aggregation followed injection of these agonists in the rat.

For these platelet stimuli, in vivo aggregometry in rat and guinea-pig may more faithfully reflect the behaviour of human platelets than in vitro studies.
PLATELET SIZE, NOT PLATELET MASS, DETERMINES INTRINSIC KINETIC DIFFERENCES IN PLATELET RECRUITMENT INTO AGGREGATES FOR ADP, U46619, AND PAF, BUT NOT FOR RISTOCETIN
Authors: Wong Truman , Frojmovic Mony M
Published: August 2018

Previous studies of platelet aggregation using resistive counting methods (PA) have suggested a dependence on platelet size (v), but have not been evaluated for varying platelet number (No) and associated total platelet mass. Here, the relationship between v, No and function was examined in size dependent human subpopulations fractionated by counterflow centrifugation. The original platelet population and three size dependent platelet fractions were concentrated and resuspended into autologous citrated platelet poor plasma at varying No for 5 donors. The initial rate and sensitivity of PA were determined generally at 3-5 seconds following ADP/ristocetin addition. Extent of PA was determined at 10 seconds. At similar No (180 ± 50 × 103 μl), large platelets (L;v = 7.4 ± 0.3 f1; 16 ± 4% of total population) were two-fold more sensitive and more rapidly recruited into both PA and turbidometrically measured macroaggregates (TA) in response to ADP than the smallest platelets (S;v = 4.6 ± 0.4; 16 ± 5%). Aggregation kinetics and sensitivity for the mid-sized platelets (v = 5.9 ± 0.3; 31 ± 7%) were intermediate between the large (L) and small (S) platelet fractions. When platelet counts were adjusted to yield similar total platelet mass (No × v), these differences persisted for PA, but not for TA. Subsequent studies were all made for platelet suspensions at similar mass. Maximal rates of ADP-induced shape change were comparable for L vs. S platelets. Significant differences in the initial rate and maximal extent of PA between the size-dependent fractions were also seen for a stable PGH2 analogue (U46619) and platelet activating factor (PAF). Most platelets were maximally recruited into micro-aggregates (60-80% PA) for all sized fractions. Kinetics and sensitivity for ristocetin-induced agglutination were comparable between the different sized fractions. The above size-dependent differences in aggregation for physiological activators appear to arise from intrinsic membrane/cell biochemical differences, not observed for ristocetin-von Willebrand (Factor VIII)-induced agglutination.
MECHANISMS OF PLATELET AGGREGATION BY ACIDIC MUCOPOLYSACCHARIDE EXTRACTED FROM STICHOPUS JAPONICUS SELENKA IN HUMANS AND RABBITS
Authors: Li J Z , Lian E C-Y
Published: August 2018

It has been reported that acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus selenka) (SJAMP) induced the aggregation of human and animal platelets by an unknown mechanism, using platelet-rich plasma (prp) and washed human and rabbit platelets we studied the effects of storage, platelet inhibitors, and various plasmas and their fractions on SJAMP-induced platelet aggregation. we found that the lowest concentrations of SJAMP required for aggregation of human and rabbit platelets were 0.4 and 2 ug/ml respectively. The reactivity of human platelets to SJAMP decreased with time after drawing of blood; rabbit platelets did not show this phenomenon. Platelet inhibitors such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose, and EDTA inhibited by 50 to 100% the aggregation of human platelets induced by SJAMP; but these inhibitors had no effect on SJAMP-induced aggregation of rabbit platelets. Washed human and rabbit platelets were not aggregated by SJAMP. The aggregation of washed human platelets by SJAMP was restored completely by human or rabbit plasma, by human fibrinogen, or by 0 to 30% saturated ammonium sulfate fraction but not by serum. The aggregation of rabbit platelets by SJAMP could only be restored by rabbit plasma or serum, or by 50 to 60% saturated ammonium sulfate fraction. The data indicate that the mechanisms of aggregation of human and rabbit platelets by SJAMP are different. THe SJAMP-induced human platelet aggregation is dependent upon metabolism, release of ADP and the cyclooxygenase pathway requiring fibrinogen and Ca++. The aggregation of rabbit platelets induced by SJAMP is independent of metabolism, release of ADP and cyclooxygenase pathway, and does not require fibrinogen and Ca++, but needs certain protein(s) in the 50 to 60% saturated ammonium sulfate fraction of rabbit plasma.
PLATELET AGGREGATION DOES NOT CONFORM TO SIMPLE PARTICLE COLLISION THEORY
Authors: Jamaluddin Moideen P
Published: August 2018

Platelet aggregation kinetics, according to the particle collision theory, generally assumed to apply, ought to conform to a second order type of rate law. But published data on the time-course of ADP-induced single platelet recruitment into aggregates were found not to do so and to lead to abnormal second order rate constants much larger than even their theoretical upper bounds. The data were, instead, found to fit a first order type of rate law rather well with rate constants in the range of 0.04 - 0.27 s-1. These results were confirmed in our laboratory employing gelfiltered calf platelets. Thus a mechanism much more complex than hithertofore recognized, is operative. The following kinetic scheme was formulated on the basis of information gleaned from the literature.

where P is the nonaggregable, discoid platelet, A the agonist, P* an aggregable platelet form with membranous protrusions, and P** another aggregable platelet form with pseudopods. Taking into account the relative magnitudes of the k*s and assuming aggregation to be driven by hydrophobic interaction between complementary surfaces of P* and P** species, a rate equation was derived for aggregation. The kinetic scheme and the rate equation could account for the apparent first order rate law and other empirical observations in the literature.
VARIATION OF FUNCTIONAL PLATELET PARAMETERS DURING STORAGE AND DURING VENOUS OCCLUSSION MEASURED
Authors: Spannagl M , Valet G , Schramm W
Published: August 2018

Information on platelet function would be of great importance for many clinical situations in addition to platelet count and bleeding time. It was the purpose of this study to test Di0c6 (3,3-dihexyl-oxacarbocyanine: transmembrane potential), AO (acridine orange: granular content), and ADB (1,4-di-acetoxy-2,3-dicyanobenzene: intracellular esterase-activity and pH) stained platelets after 1 and 5 hours storage (anticoagulated with EDTA, Heparin and Sodium-Citrate) and 2, 6, 12 and 20 minutes after venous occlussion (immediately diluted in HEPES-Buffer (1:50)). A fresh whole blood sample diluted in Buffer served as control. All blood samples were gained from normal persons. Platelets were finally diluted 1:200 in HEPES buffered saline and stained. Cell volume, green and blue fluorescence were then measured in a Fluovo-Metricell-II flow cytometer.

- The mean platelet volume increased to 111% (1 h) and 117% (5 h) of control during storage. The volume remained stable during venous occlussion.
- The transmembrane potential (Di0c6) decreased to 52% of control after 5 hours storage. We saw an increase to 141% after 20 minutes venous occlussion.

- The granular content (A0) decreased to 81% of control during storage. There was no variation during VOT.

- Esterase activity (ADB) remained constant during storage and had the lowest coefficient of variation (CV = 51%). There was an increase to 132% after 20 minutes venous occlussion.

- We saw most increase in volume and decrease in Di0c6 and A0 dye content after storage in EDTA compared to Citrate and Heparin.The present results show that the dyes of functional platelet parameters are sensitively picked up by flow cytometry. The methodology seems attractive for clinical purposes because measurements can be performed in diluted blood samples within less than five minutes after venipuncture.
PLATELET IONIZED CALCIUM MOBILIZATION (AEQUORIN METHOD) IN PATIENTS WITH PRIMARY PLATELET DYSFUNCTION
Authors: Nagayama R , Hattori A , Fuse I , Takeshige T , Takizawa S , Shibata A
Published: August 2018

Intracellular calcium level of platelets of the patients with primary platelet dysfunction (thrombasthenia, platelet cyclo-oxygenase deficiency, familial defect of A23187 induced platelet aggregation, Hermansky-Pudlak syndrome. Bernard-Soulier syndrome, each 1 case, and other 3 cases of platelet release mechanism defect of unknown etiology) were measured with the photoprotein Aequorin according to the method by Johnson et al. The peak level and the lag time to the peak were evaluated. Activation was done by 4 or more different concentrations of either thrombin (0.125-1.0μ/ml), A23187 (0.25-2.0μM), ATA2 (0.05-0.4μM) or occasionally arachidonate (0.25-100μM).

In case of stimulation by thrombin, the maximum [Ca2+] level in thrombasthenia was much lower than those in normal. The lag time was prolonged in Bernard-Soulier syndrome. In case of stimulation by STA2 the maximum [Ca2+] level was very much lower in thrombasthenia and was lower in a familial defect of A23187-induced platelet aggregation and a case of platelet release mechanism defect than normals. In case of stimulation by A23187, the maximum [Ca2+] level was much lower in thrombasthenia and PCO deficiency and platelet release mechanism defect (2 cases) in which the lag time of A23187 induced platelet aggregation was also prolonged. In PCO deficiency, arachodonate less than μM produced a dose-dependent rise in intracellular calcium level and that (1-25μM) caused a rise of a consistent level although it didn't induce aggregation. Arachidonate (25-100M) caused both higher rise and aggregation. Mobilization was normal in response to STA2 and thrombin but decreased to A23187 in this deficiency.

These findings suggest that [Ca2+] mobilization was deteriorated by many mechanisms such as defect of PGG2H2-and Tx-forma-tion, membranous abnormality (lack of glycoproteins) and storage pool deficiency, and further that atachidonate even at low concentration may cause mobilization without conversion to PGG2H2 or Tx.
STUDIES ON PLATELET ANTIGENS AGAINST SERA FROM PATIENTS WITH ITP
Authors: Hirai K , Yasunaga K , Ryo R
Published: August 2018

Chronic idiopathic thrombocytopenic purpura (ITP) is a clinical syndrome characterized by destruction of platelets by antiplatelet antiboodies. The precise pathogenic mechanism of platelet destruction in ITP is not known, although many investigators have reported that platelet-associated IgG (PAIgG) is increased in this desease. We have evaluated PAIgG in 66 patients with ITP by a competitive solidphase microenzyme immunoassay and investigated its specificity aganist antiplatelet antibody in 24 patients with ITP by Western blotting. PAIgG values were elevated in most ITP patients with platelet counts of under 50,000/μl, but within normal range in most patients with platelet count of over 50,000/μl. PAIgG values were also elevated in ITP patients with megakaryocyte counts of over 200/μl, and within normal range in most patients with normal megakaryocyte counts. Western blotting was carried out by SDS-PAGE of whole platelet lysate or platelet membrane lysate and trasfer of the platelet fraction onto nitrocellulose strips. Bound immunoglobulins were detected with an avidin-biotin-peroxidase system. Several bands of bound immunoglobulins were detected in the whole platelet lysates of ITP patients, but most of these could not be detected in platelet membrane lysates. This finding suggests that some immunoglobulins from ITP patients may bind to cytoplasmic proteins in whole platelet lysate. These observations are consistent with the hypothesis that the pathogenesis of ITP involves an immunological mechanism.
REDUCED PLATELET THROMBOXANE FORMATION IN ACUTE THROMBOTIC THROMBOCYTOPENIC PURPURA (TP): EVIDENCE FOR AN ABNORMAL PLATELET POPULATION WITH A TRANSIENT CYCLOOXYGENASE DEFECT
Authors: Scharf R E , Wehmeier A , Schneider W
Published: August 2018

We have recently shown that alpha-granule-depleted platelets circulate in acute TTP. These platelets are hemostatically defective due to partial loss of granular constituents and/or metabolic abnormalities. To further evaluate morphometric and metabolic changes of "exhausted" platelets, we studied their volume distribution and thromboxane A2 (TXA2) formation in a 35-year-old patient with primary TTP. Platelet volume distribution in whole blood was determined by the impedance method using citrate (0.38%)/glutaraldehyde (0.125%) as anticoagulant. TXB2 production was measured radioimmunologically after incubation of platelet-rich plasma (PRP) with thrombin (10 U/ml) or arachido-nic acid (AA, 450 μM) for 5 min. During the acute phase of TTP, the thrombin-induced platelet TXB2 formation was significantly reduced (n=6, 0.32±0.08, ±SD, nmoles/109 platelets) as compared to controls (n=12, 2.7±0.9 nmoles/109 platelets, p < 0.001). Incubation of PRP with exogenous AA failed to restore normal TXB2 production in acute TTP. In addition, volume analysis showed an abnormally small-sized platelet population (modal volume < 1.5 fl) during the acute phase of TTP. Clinical remission was achieved by repeated plasmapheresis with substitution of fresh frozen plasma. Following normalization of the platelet count, platelet volume gradually increased (modal volume 3.3 fl), and the platelets recovered a normal in vitro thrombin- and AA-inducible TXA2 synthesis capacity. We conclude that (1) an abnormal (small-sized) platelet population and (2) a disorder of platelet arachidonic acid metabolism exist in acute TTP, leading to reduced TXA2 production. These changes may reflect the previous activation and consumption of platelets in vivo. The reduced platelet thromboxane synthesis is consistent with a transient cyclooxygenase defect which disappears following remission of TTP.
STORAGE OF LEUKCCYTE POOR PLATELET CONCENTRATES PREPARED FROM BUFFYCOATS IN VARIOUS TYPES OF PLASTIC BAG
Authors: Pietersz R N I , Reesink H W , Dekker W J A
Published: August 2018

Platelet concentrates (PC) prepared from buffycoats in a quadruple bag CPD-SAG M system proved to have a good survival in vivo after storage for 72 h at 22°C (Vox Sang 1985, 49: 81-85). Storage of these PC for 7 days at 22°C on a linear platelet reciprocator, was studied using 6 different types of bag made of polyvinylchloride (PVC) with either DEHP and/or TOTM as plasti-cizer. From Biotest the 76 standard PVC (n=14) and 763 thin PVC (n=16)bag, from NPBI the PSV 3277 standard PVC (n=15) and DPL-110 TOTM (n=14) and from Terumo the old (n=18) and new (n=14) molded Teruflex bags were examined.

The Biotest 763, the NPBI DPL-110 and the Terumo new molded Teruflex are recommended for platelet storage.

The plasma volumes averaged 63 ml with a range of 39 to 81 ml. Platelet concentrations were comparable in all types of bag with an overall mean of 0.89±0.33 (SD)×109 /ml. The number of contaminating leukocytes and erythrocytes never exceeded 107 per unit. The initial pH was between 6.98 and 7.19. After 7 days storage the pH in all bags remained above the critical value of 6.0 with a range of 6.56 to 7.38. No decrease of PO2 was observed during storage, on the contrary a significant (p<0.05) increase in the PSV 3277 and new molded Teruflex bags was measured.

The PCO2 diminished equally in all bags also indicating adequate gas exchange. Good morphology scores of the platelets (greater than 200) were observed in 98% of the PC after 5 day storage and in 83% after 7 days.

These data indicate that PC can be equally well be stored in normal PVC bags as in special platelet storage bags, provided that the PC are leukocyte poor.
QUANTITATION OF MICROPARTICLES IN PLATELET SUSPENSIONS BY FLOW CYTOMETRY
Authors: Miller D T , Bode A P
Published: August 2018

We have examined a platelet-poor, supernatant fraction from fresh and stored platelet suspensions with a FACS 440 (Becton-Dickinson) flow cytometer to study the distribution of small microparticles previously shown to be present in citrated plasma and serum (J. George et al., Blood 60: 834, 1982). Analysis by flow cytometry offers the advantage of discrimination of populations of particles by both light scattering and immunofluorescent properties. We found two distinctly different populations of particles: the predominant one had diameters in the range of 0.1 to 0.4um and was moderately autofluorescent (AF); the other was equally AF with particle diameters of 1.0 to 3.0um and probably included a few intact platelets. By adding a precise quantity of highly fluorescent beads of 0.9um diameter to each sample, relative concentrations of particles (small and/or large) could be quantified in platelet suspensions after various treatments using ratios of particle and bead counts. The lowest concentration of particles was found in samples from whole blood collected into CPDA-1 with PGE-1 and theophylline plus sodium azide (CPT-Az). Blood in CPDA-1 alone had twice the number of small and large particles; serum had a 20X higher particle concentration. A much larger number of particles was found in platelet concentrates (PC) stored for transfusion. Fresh PC had approx. 150X higher particle concentration than CPT-Az, rising to over 200X by the eighth day of storage at 22 C. Also, we noted a shift in distribution between particle populations in stored PC toward the larger size. The concentration of larger particles alone rose from 100X relative to CPT-Az to 350X after 8 days of storage. Similar changes in supernatant platelet factor 3 (PF3) activity were noted in stored PC in another study (A.P. Bode and D.T. Miller, Vox Sanguinis 51: 299, 1986), suggesting that supernatant PF3 activity may be related to one or the other population of particles seen by flow cytometry. This technique of examining and quantifying particles in platelet preparations by flow cytometry will facilitate and expand the characterization of platelet vesiculation and the released particles.
THE EFFECT OF FLOW ON THE INTERACTION OF ISOLATED MEGAKARYOCYTES WITH EXTRACELLULAR MATRIX
Authors: Levine R F , Eldor A , Stromberg R , Vlodavsky I , HyAm E , Koslow A R , Friedman L I
Published: August 2018

To examine the effect of flow on megakaryocytes (megas) exposed to extracellular matrix (ECM), we subjected megas on ECM to laminar flow in a chamber similar to that used to study platelets circulated over aortic subendothelium. Megas, harvested from guinea pig bone marrow by centrifugal elutriation and velocity sedimentation, were allowed to adhere to cover slips coated with ECM, which were then placed in a perfusion chamber. Medium was circulated over the megas on this surface for up to 18 hrs. The cells were examined during the experiments by phase contrast and afterwards by scanning electron microscopy. With time, many attached megas developed a single, prominent, elongated pseudopod resembling the flagelliform processes observed in situ protruding into marrow sinusoids and also observed in circulating megas. In other experiments megas were introduced into the flowing medium and exposed to new ECM-coated cover slips mounted in the chamber. At shear rates of 10-200 sec−1, megas from the flowing suspension started to adhere to the ECM within 1 to 2 minutes. The number of attached cells continued to increase for several hours. Adhesion of megas under flow to the ECM was specific, since there was no adherence to glass, to glutaraldehyde-fixed ECM coated cover slips or to endothelial cells cultured on ECM coated cover slips. With time many of the megas developed the same type of pseudopods which formed in megas attached to ECM prior to flow exposure. The responses of megas to flow over ECM illustrate two aspects of mega behavior: the acquisition, even before platelet shedding, of the adhesive capacity of circulating platelets, and the possible roles of ECM and flow as anchor and inducer of platelet shedding.
MEGAKARYOCYTIC DEVELOPMENT IN LIQUID CULTURES OF CRYOPRESERVED LEUKOCYTE STEM CELL CONCENTRATES FROM CHRONIC MYELOGENOUS LEUKEMIA PATIENTS
Authors: Berthier R , Duperray A , Valiron O , Prenant M , Newton I , Schweitzer A
Published: August 2018

The proliferation and differentiation of human megakaryocytes in liquid culture has been obtained using cryopreserved light density blood cell concentrates from chronic myelogenous leukemia (CML) patients. These cryopreserved leukocytes concentrates contain a large number of viable granulo-monocytic, erythroid and megakaryocytic committed stem cells. A high number of spontaneous megakaryocytic colonies was observed in semisolid cultures plated with the CML leukocytes concentrates. A liquid culture system using RPMI 1640 supplemented with 20% human plasma (HP) has been defined where maturing megakaryocytes make up 20 to 60% of the total cells after 14 days of incubation. The same cell suspension cultured in medium supplemented with 20% foetal calf serum (FCS) showed poor megakaryocytic cell development. The megakaryocytic nature of the cells produced in HP supplemented cultures was confirmed by cytological studies and indirect immunofluorescence labeling using monoclonal antibodies (MoAb) against membrane platelet GPIb and Ilbllla, and intracellular antigens like fibrinogen and von Willebrand factor.

Ploidy of the cultured cells was studied after labeling with propidium iodide and the DNA fluorescence determined using the fluorescence activated cell sorter (FACSIV). Peaks of 8N, 16N and 32N cells were observed from HP supplemented cultures representing about 20% of the cells reacting with a GP11b111 a MoAb, while very few cells greater than 4N were observed in FCS supplemented cultures. The megakaryocytes produced in HP cultures could be further enriched by cell sorting on the FACSIV after labeling with an anti-IIbIIIa MoAb. Depending on the initial megakaryocytic concentration of the cells cultured, one to 2 é 106 megakaryocytes per hour could be harvested. Thus, cryopreserved CML blood stem cell concentrates seem to offer a reproducible source of human megakaryocytes which retain their capacity to proliferate and differentiate in liquid cultures supplemented with human plasma. These megakaryocytes can be used for the study of platelet glycoprotein biosynthesis as well as the regulation of megakaryocytopoiesis.
METABOLISM OF ARACHIDONATE RELEASED FROM THROMBIN-STIMULATED PLATELETS TO THROMBOXANE, 12-HYDROXYHEPTADECATRIENATE AND 12-HYDROXYEICOSATETRAENATE IS REGULATED BY ALBUMIN
Authors: Broekman M J
Published: August 2018

Stimulation with high-dose thrombin of human platelets leads to release of arachidonate from phosphatidylethanolamine and phosphatidylcholine via a phosholipase A2 mechanism. Released arachidonate is subsequently reincorporated into cellular lipid, or oxygenated by cyclooxygenase or lipoxygenase of the cell which originally released the arachidonate, or of another cell in the local environment. Albumin is the major fatty acid-binding protein in plasma, where it is present at 600 uM. In addition, albumin binds and stabilizes labile eicosanoid intermediates. Albumin could therefore profoundly influence the pattern and time course of eicosanoid formation. The effects of added albumin (fatty acid-free) on accumulation of free arachidonate, measured by GLC, formation of 12-hydroxyhepta-decatrienoate (12HHT) and 12-hydroxyeicosatetraenoate (12HETE), measured by HPLC, were studied in washed platelets stimulated with thrombin (15 U/5×109 cells). Addition of 150 uM albumin increased free arachidonate accumulation from 3-5 nmol to 44 nmol 300 s following thrombin stimulation. In the presence of 150 uM albumin, aspirin-pretreatment did not further increase free arachidonate accumulation. Albumin dose-dependently inhibited arachidonate cyclooxygenation: 150 uM albumin reduced 12HHT formation by >55%, but arachidonate lipoxygenation (12HETE production) decreased by only 26%. Aspirin-treated platelets, in the absence of albumin, produced more than double the quantity of 12HETE of untreated platelets. Addition of 15 uM albumin to aspirin-treated platelets increased 12HETE production slightly, but higher levels of albumin (150 uM) led to a 60% decrease in 12HETE formation. This decrease was similar to the decrease in 12HHT induced by albumin in aspirin-free platelets. Albumin also increased the lag in onset of 12HETE formation caused by aspirin. These data demonstrate that eicosanoid production by thrombin-stimulated platelets is greatly down-regulated by comparatively very small quantities of albumin due to sequestration of released arachidonate.
ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE
Authors: Purdon A D , Smith J B
Published: August 2018

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.