Protease-Activated Receptor 3 Negatively Regulates Plasmin-Mediated Protease-Activated Receptor 4 Activation in Platelets.

Abstract Plasmin, a major extracellular protease, causes intracellular signals to mediate platelet aggregation. Previously, we reported that plasmin-mediated platelet aggregation predominantly occurs through proteolytic cleavage of protease-activated receptor 4 (Quinton et al J. Biol. Chem 2004). We showed that plasmin caused aggregation of mouse platelets more readily than human platelets. In this study, we investigated the mechanism of such a differential sensitivity of mouse platelets to plasmin, using transfected cell lines and platelets. In the platelet system, plasmin caused both human and mouse platelets to shape change and aggregate in a concentration-dependent manner with a different efficiency. Whereas 0.1 U/ml of plasmin causes full aggregation of mouse platelets, that dose only induces shape change in human platelets. In transfected COS7 cells, 1 U/ml plasmin caused a higher intracellular calcium mobilization through mouse PAR4 (mPAR4) than human PAR4 (hPAR4) activation. These results indicate that the mPAR4 is primarily more readily activated by plasmin than hPAR4, possibly due to differences in the primary sequence. Further, mouse and human platelets also differ in terms of the expression of PAR3. It is known that PAR3 acts as a co-receptor for thrombin-induced PAR4 activation. In order to evaluate the contribution of mouse PAR3 to plasmin-mediated mPAR4 activation, we co-expressed mPAR3 and mPAR4 in COS7 cells. Plasmin caused lower mobilization of intracellular calcium when mPAR3 and mPAR4 are co-expressed, compared to the expression of mPAR4 alone. These results indicate that mPAR3, instead of acting as a cofactor to potentiate mPAR4 activation, inhibits plasmin-mediated mPAR4 activation. Consistent with these results, PAR3 null mouse platelets also show a greater plasmin-induced calcium mobilization and aggregation compared to wild-type mouse platelets, which express PAR3 and PAR4. In conclusion, mouse platelets are more readily activated by plasmin than human platelets due to differences in the primary sequence of PAR4. In addition, mPAR3 acts as an inhibitory receptor to plasmin-mediated PAR4 activation, instead of acting as a co-receptor.

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Negative Regulation of Gq-Mediated Pathways in Platelets by G12/13 Pathways through Fyn Kinase

Blood ◽

10.1182/blood.v112.11.2854.2854 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2854-2854

Author(s):

Soochong Kim ◽

Satya P. Kunapuli

Keyword(s):

Platelet Aggregation ◽

Intracellular Calcium ◽

Signaling Pathways ◽

Platelet Function ◽

Shape Change ◽

Wild Type Mouse ◽

Calcium Mobilization ◽

Negative Regulator ◽

Platelet Response ◽

Calcium Response

Abstract Platelets contain high levels of Src family kinases (SFKs) suggesting an important role for these enzymes in platelet function. In this study, we have investigated the regulation of platelet function by SFKs downstream of G12/13 pathways. Calcium-independent platelet shape change induced by selective G12/13 stimulation with YFLLRNP was potentiated with a small mobilization of intracellular calcium in the presence of SFK inhibitors PP1 or PP2, which was abolished by the chelation of intracellular calcium, suggesting that SFKs downstream of G12/13 negatively regulate calcium mobilization in platelets. In addition, PP1 or PP2 caused a leftward shift of human platelet aggregation, secretion, and calcium response induced by low concentrations of agonists that activate platelets through G12/13 signaling such as PAR1-activating peptide SFLLRN and PAR4-activating peptide AYPGKF. However, 2-MeSADP-induced calcium response and platelet aggregation were not affected by the presence of PP1 or PP2, suggesting that SFKs downstream of G12/13, but not Gq/Gi, pathways are involved in this platelet response. Moreover, platelet aggregation and secretion caused by combined stimulation of G12/13 and Gi/Gz (YFLLRNP + 2-MeSADP with P2Y1 antagonist/epinephrine) were also potentiated in the presence of PP1 or PP2 confirming the contribution of SFKs downstream of G12/13 as a negative regulator to platelet activation. Potentiation of platelet aggregation in the presence of SFK inhibitors was not affected in the presence of GF109203X, while PP2 failed to potentiate platelet aggregation in the presence of 5,5′-dimethyl-BAPTA indicating that potentiation of cytosolic calcium may have an important role in this enhanced platelet responses by SFK inhibition. Moreover, PP1 or PP2 failed to potentiate platelet responses in the presence of Gq selective inhibitor YM-254890 or in Gq-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of Gq signaling pathways. Importantly, AYPGKF-induced platelet aggregation, secretion, and calcium response were potentiated in Fyn-, but not in Lyn-, deficient platelets compared to the wild-type mouse platelets, whereas 2-MeSADP-induced platelet response was not affected in these platelets. We conclude that SFKs activated downstream of G12/13, but not Gq/Gi, pathways negatively regulate platelet responses by inhibiting intracellular calcium mobilization in platelets through Gq signaling pathways. Specifically, we define that Fyn plays a major role in this negatively regulatory pathway.

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Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.

Blood ◽

10.1182/blood.v114.22.770.770 ◽

2009 ◽

Vol 114 (22) ◽

pp. 770-770 ◽

Cited By ~ 2

Author(s):

Yingying Mao ◽

Todd M Getz ◽

Jianguo Jin ◽

Satya P. Kunapuli

Keyword(s):

Intracellular Calcium ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Shape Change ◽

Coagulation Factor ◽

Calcium Mobilization ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Factor Xiia

Abstract Abstract 770 Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt. Disclosures: No relevant conflicts of interest to declare.

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Evidence for Two Distinct G-protein-coupled ADP Receptors Mediating Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614427 ◽

1999 ◽

Vol 81 (01) ◽

pp. 111-117 ◽

Cited By ~ 88

Author(s):

Laurent Gousset ◽

Vinay Bhaskar ◽

Diana Vincent ◽

Albert Tai ◽

Elwood Reynolds ◽

...

Keyword(s):

Platelet Aggregation ◽

Intracellular Calcium ◽

G Protein ◽

Platelet Activation ◽

Shape Change ◽

Calcium Mobilization ◽

Intracellular Calcium Mobilization ◽

Adp Receptors ◽

Adp Receptor ◽

G Protein Coupled

SummaryThe identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTP γS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2Y1receptor. By contrast, the P2Y1receptor antagonist adeno-sine 3’,5’-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Giproteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1and coupled to Gq.

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The Gas6 Receptor Axl Enhances Platelet Activation Responses through Stimulation of PI3kinase- and PLC-Dependent Signaling Pathways.

Blood ◽

10.1182/blood.v104.11.630.630 ◽

2004 ◽

Vol 104 (11) ◽

pp. 630-630

Author(s):

Weston R. Gould ◽

Sangita Baxi ◽

Lisa A. Perrin ◽

Robert J. Leadley

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Intracellular Signaling ◽

Human Platelets ◽

Dense Granule ◽

Calcium Mobilization ◽

Dependent Manner ◽

Induced Aggregation ◽

Granule Release ◽

Stimulation Of

Abstract At the site of vascular injury, platelet activation is paramount in supporting formation of a platelet plug and generating a functional surface for the protein elements of coagulation. Recently, we demonstrated that the receptors for the α-granule constituent Gas6, support and enhance platelet aggregation and dense-granule release. The current study examined additional affects of Gas6 signaling in human platelets and sought to decipher intracellular signaling mechanisms initiated by stimulation of Axl, a Gas6 platelet receptor. Flow cytometry analyses indicated that all three Gas6 receptors, Axl, Sky, and Mer were present on the platelet surface. Blockade of Gas6, Sky, or Mer by specific antibodies not only inhibited TRAP- and ADP-induced platelet aggregation and dense granule release, but also prevented thrombin mediated clot retraction by as much as 55%. Furthermore, intracellular calcium mobilization in response to TRAP activation was greater than 80% inhibited in the presence of each of these blocking antibodies. A highly specific antibody directed toward Axl (< 2% cross reactivity with Sky and Mer) activated Axl leading to an enhancement of TRAP and ADP induced aggregation and degranulation. Stimulation of human platelets by this Axl agonist led to a modest, but sustained increase in calcium mobilization suggesting that Axl signaling incorporated activation of PLC. The increase in calcium mobilization was sensitive to wortmannin, demonstrating that PLC activation occurred concurrent with or downstream of PI3K. Indeed, additional experiments to ascertain the intracellular mediators of Axl activity identified a two-fold increase in specific phosphorylation of Akt downstream of PI3K as well as a similar increase in phosphorylation of PLCγ. TRAP stimulation of human platelets also increased the phosphorylation levels of Akt and PLCγ in a Gas6 dependent manner as a Gas6 blocking antibody reduced the levels of Akt and PLCγ phosphorylation by 50%. Overall, these studies suggest that Gas6 enhancement of human platelet activation occurs through the low-level stimulation of the intracellular signaling molecules Akt and PLCγ, serving at the juncture of several mediators of platelet activation. These events also increase levels of cytoplasmic calcium, further supporting an enhancement of activation observed in response to low levels of known platelet agonists. Thus, platelet Gas6 functions to support platelet activation at the very early stages of the hemostatic response to injury.

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Fibrinogen Binding Is Independent of an Increase in Intracellular Calcium Concentration in Thrombin Degranulated Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653769 ◽

1995 ◽

Vol 73 (02) ◽

pp. 304-308 ◽

Cited By ~ 4

Author(s):

Fabio M Pulcinelli ◽

James L Daniel ◽

Silvia Riondino ◽

Pier Paolo Gazzaniga ◽

Leon Salganicoff

Keyword(s):

Platelet Aggregation ◽

Intracellular Calcium ◽

Binding Sites ◽

Calcium Concentration ◽

Calcium Release ◽

Shape Change ◽

Cytosolic Calcium ◽

Calcium Mobilization ◽

Induce Platelet Aggregation ◽

Fibrinogen Binding

SummaryIn a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C(PLC) activation and calcium mobilization in TDP activated by ADP, epinephrine and U46619.ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

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Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645981 ◽

1987 ◽

Vol 58 (02) ◽

pp. 737-743 ◽

Cited By ~ 12

Author(s):

Frarnçois Lanza ◽

Alain Beretz ◽

Martial Kubina ◽

Jean-Pierre Cazenave

Keyword(s):

Intracellular Calcium ◽

Shape Change ◽

Calcium Ionophore ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Free Calcium ◽

Membrane Bilayer ◽

Fluorescent Indicators ◽

Fura 2 ◽

Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

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Reaction of Acetaldehyde with Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648393 ◽

1992 ◽

Vol 67 (01) ◽

pp. 126-130 ◽

Cited By ~ 8

Author(s):

Olivier Spertini ◽

Jacques Hauert ◽

Fedor Bachmann

Keyword(s):

Platelet Aggregation ◽

Inhibitory Action ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Shape Change ◽

Reaction Medium ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

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Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651597 ◽

1993 ◽

Vol 69 (03) ◽

pp. 286-292 ◽

Cited By ~ 13

Author(s):

Che-Ming Teng ◽

Feng-Nien Ko ◽

Inn-Ho Tsai ◽

Man-Ling Hung ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Inositol Phosphate ◽

Shape Change ◽

Platelet Activating Factor ◽

Atp Release ◽

Platelet Membrane ◽

Thromboxane B2 ◽

Dependent Manner ◽

Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets

Blood ◽

10.1182/blood.v77.3.500.bloodjournal773500 ◽

1991 ◽

Vol 77 (3) ◽

pp. 500-507 ◽

Cited By ~ 2

Author(s):

RN Puri ◽

F Zhou ◽

CJ Hu ◽

RF Colman ◽

RW Colman

Keyword(s):

Molecular Weight ◽

Platelet Aggregation ◽

Plasma Concentration ◽

Human Plasma ◽

Shape Change ◽

Dependent Manner ◽

Normal Human Plasma ◽

High Molecular Weight Kininogen ◽

Normal Human ◽

Dose Dependent

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)- induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin- induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma- thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma- thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

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The Role Of Calcium In Platelet Microtubule Assembly- Disassembly

10.1055/s-0038-1652479 ◽

1981 ◽

Author(s):

M Kikuchi ◽

Y Ikeda ◽

M Handa ◽

S Matsuda ◽

H Muraki ◽

...

Keyword(s):

Intracellular Calcium ◽

Dynamic Equilibrium ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Microtubule Assembly ◽

Base Line ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Transient Decrease

Microtubules exist in a dynamic equilibrium between polymerized and depolymerized forms in human platelets, playing a major role to maintain the discoid shape of platelets. It has been previously shown that the interaction of aggregating agents with platelets leads to a rapid but transient disassembly of microtubules. ( Steiner and Ikeda, J.Clin. Invest. 63:443,1979 ) In this paper, the role of calcium in the equilibrium between assembled and disassembled microtubules was investigated. The respective pools of soluble and polymerized tubulin were “frozen” by addition of a glycerol-dimethyl sulfoxide-containing medium to platelet rich plasma, preincubated with 2 µM A23187 for various time intervals. The two pools of tubulin were estimated by measuring the colchicine binding activities of total and polymerized tubulin according to the method of Wilson.Resting platelets were found to contain 56.2 ± 2.7 µg tubulin per 109 platelets, of which 56.7 % was in polymerized form. Addition of A23187 to platelet rich plasma produced a transient decrease in the pool of polymerized tubulin within 30 sec., followed by a return to base-line values within 2 min.. TMB-8, a known intracellular calcium antagonist, abolished this transient decrease in polymerized tubulin induced by A23187 in a concentration dependent manner, while indomethacin or acetylsalycylic acid did not.These findings may indicate the important role of intracellular calcium in microtubule assembly-disassembly.

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