Adrenaline Activates Human Platelets But Does Not Cause Primary Aggregation If Thrombin Generation Is Inhibited By Hirudin

1981 ◽  
Author(s):  
J P Cazenave ◽  
A Sutter ◽  
S Hemmendinger ◽  
M L Wiesel ◽  
F Lanza ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Thrombin Generation ◽  
Shape Change ◽  
Specific Inhibitor ◽  
Human Platelets

Adrenaline(ADR) affects human platelets(PLAT) by binding to a receptors,inhibiting adenylate cyclase and translocating Ca2+ across the plasma membrane. In citrated PLAT-rich plasma(CIT-PRP), ADR induces primary aggregation(1stAGG), which may be followed by 2ndAGG due to the activation of the arachidonate(AA) pathway and the release(REL) of ADP. ADR acts synergistically with other aggregating agents. In contrast, ADR does not aggregate suspensions of washed human PLAT(SWHP) and hirudin(HIR)-PRP. To determine the role of traces of thrombin(THR) on AGG and REL of 14C-5HT of prelabeled PLAT induced by ADR,we have used SWHP and PRP anticoagulated with CIT(13mM), HIR(30 U./ml) or both and examined the effects of addition of HIR(30 U./ml),a specific inhibitor of THR. ADR(1-10μM) does not cause AGG or REL of SWHP,even in the presence of added HIR or CIT. However, ADR(1-10μM) potentiates the effects of ADP and AA on AGG and REL. This is not inhibited by HIR. THR alone (0.02 U./ml) causes shape change of SWHP,addition of ADR(4.5μM) causes extensive AGG and REL, which are inhibited by HIR.In CIT-PRP, ADR(1-10μM)causes 1st AGG followed by 2ndAGG and REL, addition of HIR to CIT-PRP has no effect on AGG and REL. Aspirin(ASA) and/or CP/CPK inhibit 2ndAGG-induced by ADR in CIT-PRP. Addition of ASA+CP/CPK+HIR does not inhibit ADR-induced 1stAGG in CIT-PRP. After addition of CIT to HIR-PRP, ADR does not AGG PLAT. In contrast, ADP causes 1stAGG without REL in HIR-PRP and addition of CIT to HIR-PRP induces 2ndAGG and REL. However, ADR causes 1stAGG and REL, if PRP was prepared from blood to which HIR has been added at the same time of CIT or later. In conclusion: 1) ADR does not cause 1stAGG in SWHP or HIR-PRP if the PLAT have not been exposed to THR during their isolation; 2) traces of THR change the response of PLAT to ADR independently of the effect of CIT on Ca2+ concentrations; 3) the use of CIT-PRP does not prevent completely THR generation.

The Role Of Membrane-Bound Tubulin In Platelet Functions

1981 ◽  
Author(s):  
Y Ikeda ◽  
M Handa ◽  
Y Yoshii ◽  
M Imai ◽  
K Sugiura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Shape Change ◽  
Human Platelets ◽  
Binding Activity ◽  
Serotonin Release ◽  
Platelet Membranes ◽  
Coomassie Blue ◽  
Tubulin Subunit ◽  
Membrane Bound

Evidence has been presented which suggests the existence of tubulin, subunit protein of microtubules, as an integral part of plasma membrane of certain cells. We have investigated whether tubulin is also a constituent of platelet plasma membrane or not, and, if so, what the functional significance is? Platelet membranes isolated by glycerol lysis technique according to the method of Barber and Jamieson retained colchicine-binding activity, 6.2 ± 1.4 n mol colchicine per 100 mg platelet membranes. Colchicine-binding activity of platelet membranes was not decreased after membranes were washed 3 times, indicating that colchicinebinding activity of membranes is not due to contamination of loosely bound cytoplasmic soluble tubulin. On SDS-poly- acrylamide gel electrophoresis, platelet membranes revealed Coomassie blue stained band of molecular weight 55,000, which comigrated with purified cytoplasmic tubulin isolated from human platelets by two successive cycles of temperature -dependent polymerization depolymerization as described previously(Ikeda & Steiner, J. Biol. Chem. 251:6135, 1976). Monospecific antibody against platelet tubulin was prepared in rabbits by injecting soluble tubulin at weekly intervals for 4 weeks. Platelets preincubated with anti-tubulin F(ab’)2 fragment showed reduced platelet aggregation and shape change induced by collagen, but not by ADP or epinephrine. Collagen-induced release of 14C-serotonin was also inhibited by anti-tubulin F(ab’)2 fragment while ADP- or epinephrine-induced serotonin release was not inhibited(collagen 2μg/ml:45.6% of control, ADP 10μM:92.0% of control, epinephrine 4μg/ml: 98.0% of control).Our results suggest that membrane-associated tubulin may play important roles in collagen-platelet interactions.

scholarly journals The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin

1984 ◽  
Vol 221 (3) ◽  
pp. 897-901 ◽  
Author(s):  
T J Hallam ◽  
N T Thompson ◽  
M C Scrutton ◽  
T J Rink
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Calcium ◽  
Similar Time ◽  
Transient Rise ◽  
Thromboxane Production

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.

ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)

1987 ◽  
Author(s):  
Y Patel ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Synergistic Effects ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Presence And Absence ◽  
Pre Treatment ◽  
Arachidonate Release

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.

scholarly journals Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets

1982 ◽  
Vol 206 (1) ◽  
pp. 97-102 ◽  
Author(s):  
P Thams ◽  
K Capito ◽  
C J Hedeskov
Keyword(s):  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Specific Inhibitor ◽  
Particulate Fraction ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Rat Islets ◽  
Rat Pancreatic Islets ◽  
Mouse Islets

The effects of Ca2+-calmodulin on adenylate cyclase activity in EGTA-washed, 27000 g particulate fractions of mouse and rat pancreatic islets were studied. Ca2+ (10 microM)-calmodulin (1 microM) stimulated adenylate cyclase activity 53.1 +/- 5.2 (N = 6)% in the particulate fraction of rat islets. Trifluoperazine (50 microM), a specific inhibitor of calmodulin, inhibited the Ca2+-calmodulin activation of the adenylate cyclase activity of this fraction of rat islets. These results confirm previous reports dealing with Ca2+-Calmodulin and rat islet adenylate cyclase [Valverde, Vandermeers. Anjaneyulu & Malaisse (1979) Science 206, 225-227; Sharp, Wiedenkeller, Kaelin, Siegel & Wollheim (1980) Diabetes 29, 74-77]. In contrast, however, Ca2+ (1-100 microM)-calmodulin (1-10 microM) did not stimulate the adenylate cyclase activity in the EGTA-washed particulate fraction of mouse islets, and trifluoperazine (50 microM) did not inhibit the adenylate cyclase activity of this fraction of mouse islets, although some remaining calmodulin [0.18 +/- 0.05 (n = 3) microgram/mg of protein] could be demonstrated. GTP (10 microM) enhanced islet adenylate cyclase activity considerably, but did not confer any sensitivity towards Ca2+-calmodulin on mouse islet adenylate cyclase. The results question the role of calmodulin in the Ca2+-dependent rise in cyclic AMP evoked by glucose in pancreatic islets.

scholarly journals Phosphorylation-dependent and -independent pathways of platelet aggregation

1989 ◽  
Vol 258 (2) ◽  
pp. 479-485 ◽  
Author(s):  
S P Watson ◽  
S Hambleton
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activating Factor ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Signalling Pathways ◽  
Ionophore A23187 ◽  
Phorbol Dibutyrate ◽  
Kinase C ◽  
Dependent Pathway

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

EARLY (< 5 SEC) PHOSPHORYLATIONS OF PLATELET PROTEINS FOLLOWING ACTIVATION BY ADP AND ADRENALIN, SEPARATELY AND IN COMBINATION

1987 ◽  
Author(s):  
LR A Gear ◽  
D Freas ◽  
J D Carty
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Human Platelets ◽  
Single Particles ◽  
Specific Analysis ◽  
Mlc Phosphorylation

Understanding the earliest events (< 1 sec) in signal transduction of platelets is important, since there is evicenee that “shape change,” aggregation and secretion can all begin within this period. We have employed a guenched-flow approach to study these early events and found that thrombin can induce rapid phosphorylation of myosin light-chain kinase (20K) and a 47K protein (Blood, 67, 1738, 1986). To investigate the role of rapid phosphorylations in platelet activation, we have studied the influence of adrenalin and ADP during early (0.3 to 5 sec) stimulation. Aggregation in washed human platelets was assessed by following the loss of single particles and phosphorylation by analysing 32P-labeled proteins after gel electrophoresis. 15 µM adrenalin (without ADP) did not initiate significant aggregation or phosphorylation of myosin light chain (MLC). Phosphorylation of the 47K protein was increased by 20% at 5 sec. 0.5 µM ADP did not induce significant aggregation, but increased phosphorylation of MLC by 130% and the 47 protein by 20%. The combination of 0.5 µM ADP and 15 uM adrenalin induced significant aggregation by 0,3 sec (7.6%), which increased to 25.6% by 5 sec. Interestingly, MLC or 47K protein phosphorylation was not increased above control levels. However, the phosphorylation of four other proteins (77K, 102K, 140K and 185K), which previously had been very rapid (<1 sec) and reversible with 0.5 µM ADP alone, was now maintained, peaking at 3 sec. 10 µM ADP caused small sustained increases in phosphorylation of the same proteins. Adrenalin also caused rapid increases in the phosphorylation of 27K, 213 and 250K proteins. High levels of ADP (10 µM) only increased the 213 and 250K proteins; therefore the 27K protein appears adrenalin specific. Analysis of these early platelet phosphorylations will help understand how they are linked to initiation and maintenance of aggregation. Supported by NIH HL-27014.

scholarly journals Wortmannln, a specific inhibitor of myosin light chain kinase, prevents the agonist-stimulated plasma membrane Ca2+ entry in human platelets.

1991 ◽  
Vol 55 ◽  
pp. 91 ◽  
Author(s):  
Yoshiaki Hashimoto ◽  
Akifumi Ogihara ◽  
Kazuyuki Tokunaga ◽  
Satoshi Nakanishi ◽  
Yuzuru Matsuda ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Specific Inhibitor ◽  
Human Platelets

scholarly journals Functional enhancement of platelet activation and aggregation by erythrocytes: role of red cells in thrombosis

2014 ◽  
Author(s):  
Gabrielle E Brown ◽  
Leslie S. Ritter ◽  
Paul F. McDonagh ◽  
Zoe Cohen
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombin Generation ◽  
Splenic Macrophage ◽  
Prothrombinase Complex ◽  
Impedance Aggregometry ◽  
Increase Platelet Aggregation ◽  
Experimental Group

Platelets expose phosphatidylserine (PS), a component of the prothrombinase complex, on the outer surface of the plasma membrane when activated. [ref 1] The prothrombinase complex catalyzes the conversion of prothrombin to thrombin, and it has been demonstrated that an increase in PS exposure is correlated with an increase in thrombin generation by platelets. [refs 2,3] Similarly, erythrocyte (RBC) activation, or eryptosis, is also characterized by PS exposure on the plasma membrane. [ref 4] Although PS exposure on RBCs is considered a signal for splenic macrophage destruction, eryptosis may allow RBCs to contribute to thrombosis.[ref 4] The aims of this study were to determine whether the addition of RBCs to platelets increased functional platelet aggregation and coagulation properties. A ratio of 4 RBCs to 1 platelet (4:1) was evaluated for aggregation and coagulation compared to platelet control. Platelet aggregation and coagulation properties were evaluated with impedance aggregometry and thromboelastography, respectively. The 4:1 experimental group had significant increases in aggregation and coagulation relative to the platelet control. These results indicate that RBCs increase platelet aggregation and coagulation properties. This suggests that RBCs play a role in diseases traditionally thought of as associated solely via dysregulated platelet activation.

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