scholarly journals Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets

Molecules ◽  
2021 ◽  
Vol 26 (17) ◽  
pp. 5340
Author(s):  
Li Ming Lien ◽  
Wan Jung Lu ◽  
Kuan Hung Lin ◽  
Ling Hsuan Kang ◽  
Ting Yu Chen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Immune Thrombocytopenia ◽  
Antiplatelet Agents ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Glycoprotein Vi ◽  
Mitogen Activated Protein ◽  
Normal Hemostasis

Vincristine is a clinically used antimicrotubule drug for treating patients with lymphoma. Due to its property of increasing platelet counts, vincristine is also used to treat patients with immune thrombocytopenia. Moreover, antiplatelet agents were reported to be beneficial in thrombotic thrombocytopenic purpura (TTP). Therefore, we investigated the detailed mechanisms underlying the antiplatelet effect of vincristine. Our results revealed that vincristine inhibited platelet aggregation induced by collagen, but not by thrombin, arachidonic acid, and the thromboxane A2 analog U46619, suggesting that vincristine exerts higher inhibitory effects on collagen-mediated platelet aggregation. Vincristine also reduced collagen-mediated platelet granule release and calcium mobilization. In addition, vincristine inhibited glycoprotein VI (GPVI) signaling, including Syk, phospholipase Cγ2, protein kinase C, Akt, and mitogen-activated protein kinases. In addition, the in vitro PFA-100 assay revealed that vincristine did not prolong the closure time, and the in vivo study tail bleeding assay showed that vincristine did not prolong the tail bleeding time; both findings suggested that vincristine may not affect normal hemostasis. In conclusion, we demonstrated that vincristine exerts antiplatelet effects at least in part through the suppression of GPVI signaling. Moreover, this property of antiplatelet activity of vincristine may provide additional benefits in the treatment of TTP.

The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

1972 ◽  
Vol 28 (01) ◽  
pp. 031-048 ◽  
Author(s):  
W. H. E Roschlau ◽  
R Gage
Keyword(s):  
Platelet Aggregation ◽  
Aspergillus Oryzae ◽  
Degradation Products ◽  
Blood Platelet ◽  
Human Platelets ◽  
Fibrinolytic Enzyme ◽  
Inhibitory Effects ◽  
Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

Defective Activation of Phospholipase Cγ2 by Collagen in Platelets Lacking the Semaphorin Family Member, Sema4D.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 417-417 ◽  
Author(s):  
Li Zhu ◽  
Hong Jiang ◽  
Atsushi Kumanogoh ◽  
Hitoshi Kikutani ◽  
Lawrence F. Brass
Keyword(s):  
Platelet Aggregation ◽  
Family Member ◽  
Neural Development ◽  
Tyrosine Phosphatase ◽  
Large Family ◽  
Arterial Injury ◽  
Human Platelets ◽  
Glycoprotein Vi

Abstract Semaphorins are a large family of cell surface molecules best known for their ability to mediate communication between cells during neural development. We have recently shown that human platelets express the semaphorin family member, sema4D, and both of its known receptors, CD72 and plexin-B1 (Zhu, et al, PNAS, 2007). We have also shown that sema4D(−/−) mice have an impaired response to arterial injury in vivo and a selective defect in collagen- and convulxin-induced platelet aggregation in vitro. In the present studies we have sought the molecular basis for these defects, focusing on events downstream of glycoprotein VI (GPVI), which serves as a receptor for both collagen and convulxin. In normal platelets, GPVI signaling leads to the phosphorylation and activation of phospholipase Cγ2 (PLCγ2) through the formation of a signaling complex that includes SLP-76 and LAT. This complex is activated when GPVI-associated FcRγ is phosphorylated, allowing the tyrosine kinase, Syk, to bind. PLCγ2 activation results in phosphoinositide hydrolysis, an IP3-mediated increase in cytosolic Ca++, and activation of additional kinases, such as Akt. In theory, the absence of sema4D could affect any of these steps and by doing so impair collagen-induced platelet aggregation. Working backwards through the GPVI pathway, our results showed that compared to platelets from matched WT mice, sema4D(−/−) platelets have 1) a rightward-shift in the dose/response curve for collagen-induced Akt phosphorylation, 2) a 37% smaller increase in cytosolic Ca++, and 3) a 43% smaller increase in PLCγ2 phosphorylation. However, we found no defect in collagen-induced FcRγ phosphorylation, which is the earliest event in GPVI signaling. The defect in PLCγ2 phosphorylation was not limited to mouse platelets, but was also observed when human platelets were stimulated with collagen in the presence of an antibody directed towards the sema4D extracellular domain. Taken together, these results show that sema4D is needed for optimal activation of PLCγ2 by collagen downstream of the GPVI/FcRγ complex. Sema4D is believed to act in part through contact-dependent binding of sema4D to its receptors, CD72 and plexin-B1. Since these studies were performed under conditions in which platelet:platelet contacts can occur, the observed defect in collagen and convulxin responses could be due to impaired signaling by either of these receptors or, in theory, by retrograde signaling via sema4D. One candidate mechanism involves a regulatory complex between CD72 and the tyrosine phosphatase, SHP-1, which we have shown to occur in resting human platelets and to be lost when platelets are activated by agonists or stimulated by soluble sema4D. In theory, sema4D-dependent loss of the CD72/SHP-1 complex allows SHP-1 to relax into an inactive conformation, promoting protein tyrosine phosphorylation, which would not occur when sema4D is absent or blocked.

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

Circulation ◽  
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.

Arginine vasopressin release from human platelets after irreversible aggregation

1990 ◽  
Vol 78 (1) ◽  
pp. 113-116 ◽  
Author(s):  
Giovanni Anfossi ◽  
Elena Mularoni ◽  
Mariella Trovati ◽  
Paola Massucco ◽  
Luigi Mattiello ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Arginine Vasopressin ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Vasopressin Release ◽  
Irreversible Aggregation ◽  
Local Release

1. The release of arginine vasopressin from human platelets was investigated in platelet-rich plasma after irreversible aggregation induced by adenosine 5′-pyrophosphate, collagen, sodium arachidonate, thrombin and adrenaline in vitro. 2. Arginine vasopressin levels were significantly higher in the supernatant from stimulated platelet-rich plasma than from unstimulated samples, reaching 3.5 × 10−12 (range 1.6–12.5 × 10−12) mol/l in the absence of an aggregating agent, 8.8 × 10−12 (range 4.2–17.5 × 10−12) mol/l after adenosine 5′-pyrophosphate, 13.7 × 10−12 (2.2–63.2 × 10−12) mol/l after collagen, 7.8 × 10−12 (2.2–14.6 × 10−12) mol/l after sodium arachidonate, 7.8 × 10−12 (2.2–16.3 × 10−12) mol/l after thrombin and 12.2 × 10−12 (4.8–32.1 × 10−12) mol/l after adrenaline. 3. An arginine vasopressin level of 18 × 10−12 mol/l, which can be achieved physiologically, increased the sensitivity of platelets to adenosine 5′-pyrophosphate and collagen in vitro; the same concentration of arginine vasopressin caused a potentiation of the effect of catecholamines on the response of platelets to sodium arachidonate. 4. These results indicate that intraplatelet arginine vasopressin is released during aggregation and suggest that a local release of arginine vasopressin could occur after complete platelet aggregation in vivo.

scholarly journals Survival and recovery of human platelets stored for five days in a non- plasma medium

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 672-675 ◽  
Author(s):  
GA Adams ◽  
SD Swenson ◽  
G Rock
Keyword(s):  
Platelet Aggregation ◽  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Plasma Medium ◽  
Release Reaction ◽  
In Vivo Recovery ◽  
Human Blood Platelets

Abstract Human blood platelets were stored for five days as concentrates in 60 mL of: (a) plasma; (b) non-plasma medium with anticoagulant; and (c) non-plasma medium without anticoagulant. All preparations were equally functional when tested for platelet aggregation and release reaction in response to single agonist or synergistic pairs of agonists in vitro. Platelets stored in non-plasma medium with anti-coagulant had lower kallikrein, fibrino(gen)peptide A, lactate, and beta-thromboglobulin than did plasma controls after five days. In vivo recovery and survival of platelets stored in non-plasma medium with anticoagulant were 51.2% +/- 4.3% and 8.7 +/- 0.3 days, respectively, which were not statistically different from plasma controls of 39.2% +/- 4.9% and 7.2 +/- 0.8 days, respectively. It is concluded that platelets can be stored for five days in a non-plasma medium and still have good in vivo recoveries and survivals.

scholarly journals Synthesis and Thrombin, Factor Xa and U46619 Inhibitory Effects of Non-amidino and Amidino N2-Thiophenecarbonyl- and N2-Tosylanthranilamides

2017 ◽  
Author(s):  
Soo Hyun Lee ◽  
Wonhwa Lee ◽  
Nguyen Thi Ha ◽  
Il Soo Um ◽  
Jong-Sup Bae ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Factor Xa ◽  
Inhibitory Effects ◽  
Human Endothelial Cells ◽  
Antithrombotic Activity ◽  
Pharmacological Targets

Thrombin (factor IIa) and factor Xa (FXa) are key enzymes at the junction of the intrinsic and extrinsic coagulation pathways and are the most attractive pharmacological targets for the development of novel anticoagulants. Twenty non-amidino N2-thiophencarbonyl- and N2-tosyl anthranilamides 1-20 and six amidino N2-thiophencarbonyl- and N2-tosylanthranilamides 21-26 were synthesized and evaluated prothrombin time (PT) and activated partial thromboplastin time (aPTT) using human plasma at concentration 30 μg/mL in vitro. From these results, compounds 5, 9, and 21-23 were selected to study the further antithrombotic activity. The anticoagulant properties of 5, 9, and 21-23 significantly exhibited a concentration-dependent prolongation of in vitro PT and aPTT, in vivo bleeding time, and ex vivo clotting time. These compounds concentration-dependently inhibited the activities of thrombin and FXa and inhibited the generation of thrombin and FXa in human endothelial cells. In addition, data showed that 5, 9, and 21-23 significantly inhibited thrombin catalyzed fibrin polymerization and mouse platelet aggregation and inhibited platelet aggregation induced U46619 in vitro and ex vivo. N-(3'-Amidinophenyl)-2-((thiophen-2''-yl)carbonyl amino)benzamide (21) was most active.

Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model

1998 ◽  
Vol 79 (01) ◽  
pp. 222-227 ◽  
Author(s):  
F. Stockmans ◽  
W. Deberdt ◽  
Å. Nyström ◽  
E. Nyström ◽  
J. M. Stassen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Peak Plasma ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Cell Deformability

SummaryIntravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.

Monosubstituted Coumarins Inhibit Epinephrine-Induced Platelet Aggregation Antiplatelet Effect of Monosubstituted Coumarins

2021 ◽  
Vol 19 ◽  
Author(s):  
Fausto Alejandro Jiménez-Orozco ◽  
Sergio Galicia-Zapatero ◽  
Edgar López-López ◽  
José L. Medina-Franco ◽  
Fernando León Cedeño ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
In Silico ◽  
Drug Targets ◽  
Platelet Reactivity ◽  
Antiplatelet Agents ◽  
Human Platelets ◽  
In Silico Studies ◽  
Vitro Effect ◽  
Induced Aggregation

Aim: Evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various pro-aggregatory agonists, particularly by epinephrine. Background: The emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited. Objective: Establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Using in silico studies, suggest potential drug targets to which the molecules bind to produce antiplatelet effects. Methods: The platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/μl) with epinephrine (10 µM), collagen (2 µg / ml) or ADP (10 µM). The aggregation of controls platelets was considered 100% of the response for each pro-aggregatory agonists. Results: Eleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer. Conclusions : In silico studies suggest that most active molecules might have antagonistic interactions in the adrenoceptors α2 and β2. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.

Effectiveness of a Novel Humanized VB22B Minibody against Human TPO Receptor Compared with Recombinant TPO and Eltrombopag Using Primary Human CD34+ Cells and Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4559-4559
Author(s):  
Eri Matsuki ◽  
Akiko Yamane ◽  
Shinichiro Okamoto ◽  
Yoshitaka Miyakawa
Keyword(s):  
Bone Marrow ◽  
Platelet Aggregation ◽  
Colony Formation ◽  
Human Platelets ◽  
Receptor Agonists ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proplatelet Formation

Abstract Abstract 4559 Thrombopoietin (TPO) is a cytokine produced primarily by the liver and kidney that regulates platelet production by stimulating proliferation and differentiation of hematopoietic stem cells, megakaryocytic progenitor cells and megakaryocytes via activation of its receptor, c-Mpl. Recently, TPO receptor agonists such as eltrombopag and romiplostim have been approved for chronic ITP. huVB22B was created as a novel humanized form of murine sc(Fv) 2VB22B minibody (BLOOD, 2005) which activates human c-Mpl by CDR grafting. The advent of these various TPO receptor agonists prompted us to consider the differences in their mechanisms of action, efficacy or potency. However, to date, there has been no in vivo or in vitro study directly comparing the effects of different TPO receptor agonists. In this study, we compared the efficacy of huVB22B on CFU-GM, CFU-E, CFU-Megakaryocyte (CFU-MK), megakaryocyte maturation (DNA ploidy and proplatelet formation) with those of recombinant human TPO (rhTPO) and eltrombopag. Primary human CD34+ bone marrow cells were cultured with various concentrations of rhTPO, huVB22B and eltrombopag using methylcellulose based media. In serum-free condition, 0.286 nM rhTPO, 0.182 nM huVB22B and 17.7 mcM eltrombopag demonstrated almost equivalent efficacy of megakaryocyte colony formation. At these concentrations, all agents demonstrated similar in vitro efficacy for colony formation of CFU-GM and CFU-E, proplatelet formation and nuclear maturation of megakaryocytes. In preliminary results, huVB22B induced maturation of CFU-MK earlier than rhTPO and eltrombopag, suggesting that huVB22B might have some potential to increase human platelets faster than other agents in vivo. This is compatible with the observation that huVB22B induced tyrosine phosphorylation of STAT3, STAT5 and JAK2 faster and stronger than rhTPO and eltrombopag in human primary platelets. Both rhTPO and huVB22B enhanced low-dose ADP and collagen-induced human platelet aggregation in vitro. In contrast, eltrombopag did not enhance ADP or collagen-induced platelet aggregation, although it induced activation of JAK-STAT pathway in human platelets. Contrary to the fact that huVB22B induces phosphorylation of intracellular signaling molecules faster and stronger than rhTPO in human platelets, the priming effect by huVB22B on platelet aggregation was much weaker than rhTPO. In conclusion, we confirmed that newly created huVB22B minibody induced colony formation of CFU-MK, CFU-E, CFU-GM and maturation of megakaryocytes from human bone marrow-derived CD34+ cells in vitro. The differences among TPO receptor agonists observed in our study would lead to further understanding of the basic biology of megakaryopoiesis and the action of TPO receptor agonists. Disclosures: Okamoto: Alexion: Research Funding. Miyakawa:GlaxoSmithKline: Consultancy.

Glycosylation of autoantibodies: Insights into the mechanisms of immune thrombocytopenia

2013 ◽  
Vol 110 (12) ◽  
pp. 1259-1266 ◽  
Author(s):  
Tamam Bakchoul ◽  
Kathrin Walek ◽  
Annika Krautwurst ◽  
Mathias Rummel ◽  
Gregor Bein ◽  
...  
Keyword(s):  
Immune Thrombocytopenia ◽  
Laser Scanning ◽  
Human Platelets ◽  
Laser Scanning Microscopy ◽  
Effector Functions ◽  
Scid Mouse Model ◽  
Rapid Clearance

SummaryImmune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets (PLTs). IgG effector functions depend on their Fc-constant region which undergoes post-translational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. AAbs were purified from ITP patients (n=15) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and activated complement. N-glycan cleavage significantly reduced both effects. In vivo survival of human PLTs was assessed after co-transfusion with native or N-glycan cleaved AAbs in a NOD/SCID mouse model. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL5h) 75% vs 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL5h 82% vs 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL5h 42%, p<0.001). N-glycosylation of human ITP AAbs appears to be required for platelet phagocytosis and complement activation, reducing platelet survival in vivo. Posttranslational modification of AAbs may constitute an important determinant for the clinical manifestation of ITP.

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