Quantification and optimization of clot retraction in washed human platelets by Sonoclot coagulation analysis

SummaryHuman platelets frozen at −195° C (liquid nitrogen) retain their morphological integrity and ability to promote clot retraction when 5% dimethyl-sulfoxide and 5% dextrose are added to the suspending plasma medium. Slow freezing was more effective than direct immersion in the liquid nitrogen. Although similar results may be achieved with dimethylsulfoxide alone with rigidly controlled freezing rates, the addition of sugars may permit freezing under less critical conditions.Dimethylsulfoxyd und 5% Dextrose dem Plasmamilieu hinzugefügt werden. Das langsame Einfrieren ist effektiver als das direkte Eintauchen in flüssigen Stickstoff. Obschon ähnliche Resultate mit Dimethylsulfoxyd allein unter exakter Kontrolle der Einfrierungsgeschwindig-keit erreicht werden können, erlaubt die Zugabe von Dextrose ein Einfrieren unter weniger kritischen Bedingungen.

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Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽

10.1161/circ.142.suppl_3.13598 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Ahmed Alarabi ◽

Zubair Karim ◽

Victoria Hinojos ◽

Patricia A Lozano ◽

Keziah Hernandez ◽

...

Keyword(s):

G Protein ◽

Platelet Function ◽

Thrombus Formation ◽

Human Platelets ◽

Inhibitory Effects ◽

Clot Retraction ◽

Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

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Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets

International Journal of Molecular Sciences ◽

10.3390/ijms20215430 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5430

Author(s):

Ildikó Beke Debreceni ◽

Gabriella Mezei ◽

Péter Batár ◽

Árpád Illés ◽

János Kappelmayer

Keyword(s):

Chronic Myeloid Leukemia ◽

Western Blot ◽

Myeloid Leukemia ◽

Thrombin Generation ◽

Platelet Adhesion ◽

Kinase Inhibitors ◽

Human Platelets ◽

Activation Loop ◽

Clot Retraction ◽

Lines Of Evidence

Tyrosine kinase inhibitors (TKI) such as the BCR-ABL inhibitor dasatinib and nilotinib are highly effective therapies for chronic myeloid leukemia (CML). However, several lines of evidence suggest that dasatinib can induce bleeding which may be due to impaired collagen-induced platelet adhesion, aggregation, and secretion. Sarcoma family kinases (SFK) play central role in the GPVI-induced signaling pathway. We aimed to investigate whether and how dasatinib can modulate SFK-mediated platelet procoagulant activity in a purified system and in dasatinib/nilotinib treated CML patients. In platelet rich plasmas of healthy volunteers, dasatinib dose-dependently reduced convulxin-induced phosphatidylserine exposure and attenuated thrombin formation. Similarly to these changes, integrin activation and clot retraction were also significantly inhibited by 100 nM dasatinib. Platelets isolated from dasatinib treated patients showed a significantly lower phosphatidylserine expression upon convulxin activation compared to premedication levels. In these samples, thrombin generation was significantly slower, and the quantity of formed thrombin was less compared to the trough sample. Western blot analyses showed decreased phosphorylation levels of the C-terminal tail and the activation loop of SFKs upon dasatinib administration. Taken together, these results suggest that dasatinib inhibits the formation of procoagulant platelets via the GPVI receptor by inhibiting phosphorylation of SFKs.

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PF801 DASATINIB ATTENUATES THROMBIN GENERATION AND CLOT RETRACTION OF CONVULXIN ACTIVATED HUMAN PLATELETS

HemaSphere ◽

10.1097/01.hs9.0000561484.73435.53 ◽

2019 ◽

Vol 3 (S1) ◽

pp. 353

Author(s):

I. Beke Debreceni ◽

M. Haddadeen ◽

J. Kappelmayer

Keyword(s):

Thrombin Generation ◽

Human Platelets ◽

Clot Retraction

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AMPK α2 subunit is involved in platelet signaling, clot retraction, and thrombus stability

Blood ◽

10.1182/blood-2010-04-279612 ◽

2010 ◽

Vol 116 (12) ◽

pp. 2134-2140 ◽

Cited By ~ 30

Author(s):

Voahanginirina Randriamboavonjy ◽

Johann Isaak ◽

Timo Frömel ◽

Benoit Viollet ◽

Beate Fisslthaler ◽

...

Keyword(s):

Kinase Inhibitors ◽

Adenosine Monophosphate ◽

Human Platelets ◽

Whole Body ◽

Initial Increase ◽

Clot Retraction ◽

Compound C ◽

Platelet Signaling ◽

Upstream Kinase

Abstract The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is a regulator of energy balance at the cellular and whole-body levels, but little is known about the role of AMPK in platelet activation. We report that both the α1 and α2 AMPK isoforms are expressed by human and murine platelets and that thrombin elicits the phosphorylation of AMPKα as well as the upstream kinase, liver kinase B1 (LKB1). In human platelets, the kinase inhibitors iodotubercidin and compound C significantly inhibited thrombin-induced platelet aggregation and clot retraction without affecting the initial increase in [Ca2+]i. Clot retraction was also impaired in platelets from AMPKα2−/− mice but not from wild-type littermates or AMPKα1−/− mice. Moreover, rebleeding was more frequent in AMPKα2−/− mice, and the FeCl3-induced thrombi formed in AMPKα2−/− mice were unstable. Mechanistically, AMPKα2 was found to phosphorylate in vitro the Src-family kinase, Fyn, and isoform deletion resulted in the attenuated threonine phosphorylation of Fyn as well as the subsequent tyrosine phosphorylation of its substrate, β3 integrin. These data indicate that AMPKα2—by affecting Fyn phosphorylation and activity—plays a key role in platelet αIIbβ3 integrin signaling, leading to clot retraction and thrombus stability.

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Inhibition of PF4 and ßTG Release from Platelets by Piracetam

10.1055/s-0039-1687600 ◽

1979 ◽

Author(s):

R.L. Henry ◽

R.M. Nalbandian ◽

G.E. Herman ◽

T. Ho

Keyword(s):

Platelet Aggregation ◽

Normal Individual ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Complete Inhibition ◽

Clot Retraction ◽

Platelet Factor ◽

Storage Pool ◽

No Inhibition ◽

No Release

Platelet factor four (PF4) and beta-thromboglobulin (βTG) were released from human platelets alpha granules by ADP and epinephrine and measured by radioimmunoassay. Both release materials are antiheparins but PF4 is reported to be more potent. However, PF4 is released at about 1/4 the level of βTG in nanog rams/ml. Total release occurred with 5 ugm/rnl ADP in platelet-rich-plasma adjusted to 200,000 platelets/mm3 and with 1.25 × 10-5M epinephrine. No further release was found by freeze-thawing procedures. In one case, no release occurred although full aggregation proceeded normally with both mediators. Only minimal amounts were recorded after freeze-thawing indicating a storage pool deficiency of PF4 and βTG in an apparantly normal individual. Complete inhibition of PF4 and βTG release was obtained concurrently with elimination of the 2nd epinephrine wave by 6.4 × 10-4 M Piracetam. In contrast to aspirin, no inhibition of ADP, Collagen, or Ristocetin aggregation or release occurred with Piracetam. In previous work it was determined that Piracetam even at 6.4 × 10-3 M did not modify thrombin, prothrombin, or activated partial thromboplastin times. In addition, clot retraction was not modified in concentrations of Piracetam as high as 1.28 × 10-2 M known to eliminate the 2nd wave of platelet aggregation by epinephrine.

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PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

Thrombosis and Haemostasis ◽

10.1160/th13-06-0484 ◽

2014 ◽

Vol 111 (03) ◽

pp. 508-517 ◽

Cited By ~ 30

Author(s):

Carol Dangelmaier ◽

Bhanu Kanth Manne ◽

Elizabetta Liverani ◽

Jianguo Jin ◽

Paul Bray ◽

...

Keyword(s):

Protein Kinase ◽

Protein A ◽

Human Platelets ◽

Clot Retraction ◽

Functional Responses ◽

Atp Secretion ◽

Dependent Protein ◽

Induced Aggregation

Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

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Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

Blood ◽

10.1182/blood.v51.6.1065.1065 ◽

1978 ◽

Vol 51 (6) ◽

pp. 1065-1071 ◽

Cited By ~ 48

Author(s):

S Levy-Toledano ◽

G Tobelem ◽

C Legrand ◽

R Bredoux ◽

L Degos ◽

...

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Shape Change ◽

Antigenic Site ◽

Human Platelets ◽

Clot Retraction ◽

Igg Antibody ◽

Normal Human ◽

Induced Aggregation ◽

Bovine Factor

Abstract In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

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Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649981 ◽

1995 ◽

Vol 74 (06) ◽

pp. 1551-1556 ◽

Cited By ~ 25

Author(s):

Lisa K Jennings ◽

Melanie M White ◽

Timothy D Mandrell

Keyword(s):

Platelet Aggregation ◽

Conformational Changes ◽

Low Dose ◽

Species Differences ◽

Receptor Occupancy ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Clot Retraction ◽

Fibrinogen Receptor ◽

Functional Aspects

SummaryWe examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

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Platelet Adhesion Plaques: Anchors for Contraction of the Hemostatic Plug.

Blood ◽

10.1182/blood.v114.22.5128.5128 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5128-5128

Author(s):

James G. White ◽

Steven M. Burris ◽

Gines Escolar

Keyword(s):

Actin Filaments ◽

Conflicts Of Interest ◽

Membrane Surface ◽

Protein A ◽

Human Platelets ◽

Contractile Force ◽

Actin Binding ◽

Clot Retraction ◽

Rhodamine Phalloidin ◽

Adhesion Plaques

Abstract Abstract 5128 Exposure of GPIIb-IIIa and other receptors on the surface of activated platelets, binding of fibrinogen, molding of shape-changed cells into tight aggregates, internal assembly of actin molecules into filaments and movement of talin, an actin-binding protein, to the inner membrane surface provides the framework for clot retraction. However, the direction of contractile force towards the center of large aggregates or clots would lift the hemostatic plug away from the edges of vascular injury. Another mechanism must be present to facilitate the direction of contractile force toward the damaged vessel wall. This may be accomplished by development of adhesion plaques as platelets spread out on the injured vessel. The present study has used scanning (SEM) and transmission electron microscopy (TEM), confocal and immunofluorescence microscopy to detect adhesion plaques developing at sites of contact as platelets spread on surfaces. Rhodamine-phalloidin was used to detect actin filaments, and an anti-talin antibody identified by protein-A gold or Alexa Fluor 488 labeled rabbit anti mouse IgG to demonstrate talin. Normal human platelets were spread on clean glass slides or plastic chambers for intervals of up to 90 min, extracted with Triton X100 or fixed intact then labeled for talin and actin, and prepared for study by the several microscopic techniques. Triton-extracted spread platelets revealed attachment plaques well stained for talin and actin when examined by SEM or TEM. Inmunofluorescence studies of spread platelets stained with rhodamine-phalloidin and antibodies also revealed co-participation of actin filaments and talin in formation of the adhesion plaques. The association of actin and talin remained intact at all intervals for up to 90 min. Clearly adhesion plaques serve as the anchors for contraction and sealing of hemostatic plug to damaged vascular surfaces. Disclosures No relevant conflicts of interest to declare.

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