scholarly journals Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo

2009 ◽  
Vol 206 (11) ◽  
pp. 2365-2379 ◽  
Author(s):  
Stefania Momi ◽  
Emanuela Falcinelli ◽  
Silvia Giannini ◽  
Loredana Ruggeri ◽  
Luca Cecchetti ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Arterial Injury ◽  
Human Platelets ◽  
Matrix Metalloproteinase 2 ◽  
Kinase Activation ◽  
A Site ◽  
Arterial Damage

Platelet activation at a site of vascular injury is essential for the arrest of bleeding; however, excessive platelet activation at a site of arterial damage can result in the unwarranted formation of arterial thrombi, precipitating acute myocardial infarction, or ischemic stroke. Activation of platelets beyond the purpose of hemostasis may occur when substances facilitating thrombus growth and stability accumulate. Human platelets contain matrix metalloproteinase 2 (MMP-2) and release it upon activation. Active MMP-2 amplifies the platelet aggregation response to several agonists by potentiating phosphatidylinositol 3-kinase activation. Using several in vivo thrombosis models, we show that the inactivation of the MMP-2 gene prevented thrombosis induced by weak, but not strong, stimuli in mice but produced only a moderate prolongation of the bleeding time. Moreover, using cross-transfusion experiments and wild-type/MMP-2−/− chimeric mice, we show that it is platelet-derived MMP-2 that facilitates thrombus formation. Finally, we show that platelets activated by a mild vascular damage induce thrombus formation at a downstream arterial injury site by releasing MMP-2. Thus, platelet-derived MMP-2 plays a crucial role in thrombus formation by amplifying the response of platelets to weak activating stimuli. These findings open new possibilities for the prevention of thrombosis by the development of MMP-2 inhibitors.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

A Humanin Analog Attenuates Platelet Responses to Vascular Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1131-1131 ◽  
Author(s):  
Lijie Ren ◽  
Qiang Li ◽  
Zhao Zeng ◽  
Peipei Mou ◽  
Xiaohui Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Ischemia Reperfusion ◽  
Treated Group ◽  
Human Platelets ◽  
Control Group ◽  
Arterial Thrombus ◽  
Group 3

Abstract Abstract 1131 Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P<0.05) and 71.9% (P<0.001), respectively. Similarly, HNG significantly reduced ATP release stimulated by collagen or convulxin. Convulxin-induced P-selectin expression and fibrinogen binding on single platelet was inhibited by HNG, as measured by flow cytometry. Moreover, HNG reduced platelet spreading on the fibrinogen coated surface by 62.9 % (P <0.05). Western blot revealed a reduction of platelet AKT phosphorylation by HNG upon collagen stimulation, implying the involvement of PI3K pathway. In addition, MAPK P38 phosphorylation by collagen and convulxin was also reduced by HNG. HNG effects on thrombus formation were tested in vivo in a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of HNG (25μg/kg) to male C57BL6/J mice significantly extended the first occlusion time (7.3±0.4 min, N=10), when compared to the saline injected littermates (5.4±0.7 min, N=12) (P <0.05). Furthermore, the number of mice that formed stable thrombus was less in the HNG–treated group (3/13) than the control group (6/13), while the non-occlusion mouse number was more in the HNG-treated group (3/13) than the control group (1/13). Together, these data show that HNG inhibits platelet activation and arterial thrombus formation. This might suggest that the protective effects of HNG against ischemia reperfusion injury could be, in part, via attenuating platelet activation. Therefore, HNG could be a potential therapeutic agent in thrombotic and cardiovascular disorders. Disclosures: No relevant conflicts of interest to declare.

Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions

2016 ◽  
Vol 115 (02) ◽  
pp. 333-343 ◽  
Author(s):  
Stefania Momi ◽  
Philip G. de Groot ◽  
Monica Battiston ◽  
Luigi de Marco ◽  
Emanuela Falcinelli ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Platelet Activation ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Vascular Wall ◽  
Matrix Metalloproteinase 2 ◽  
High Shear ◽  
Flow Conditions ◽  
Filtration System ◽  
Platelet Deposition

SummaryPlatelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O’Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i. e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, p< 0.05) and increased retained platelets (from 72.3 ± 2.3 % to 81.1 ± 1.8 %, p< 0.05) in the O’Brien system, an effect prevented by a specific MMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec-1) (maximum +47.0 ± 11.9 %, p< 0.05, with 50 ng/ml), while pre-incubation with a MMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0 % vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

2018 ◽  
Vol 7 (11) ◽  
pp. 440 ◽  
Author(s):  
Wan Lu ◽  
Chi Chung ◽  
Ray Chen ◽  
Li Huang ◽  
Li Lien ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Phospholipase D ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Activity ◽  
Clot Retraction ◽  
First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

Triggering Receptor Expressed on Myeloid cells-1: a new player in platelet aggregation

2017 ◽  
Vol 117 (09) ◽  
pp. 1772-1781 ◽  
Author(s):  
Lucie Jolly ◽  
Jérémie Lemarié ◽  
Kevin Carrasco ◽  
Batric Popovic ◽  
Marc Derive ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Inflammatory Diseases ◽  
Thrombus Formation ◽  
Myeloid Cells ◽  
Human Platelets ◽  
Excessive Bleeding ◽  
Supplementary Material ◽  
Pharmacologic Inhibition

SummaryTriggering Receptor Expressed on Myeloid cells-1 (TREM-1) is an immunoreceptor initially known to be expressed on neutrophils and monocytes/macrophages. TREM-1 acts as an amplifier of the inflammatory response during both infectious and aseptic inflammatory diseases. Another member of the TREM family, The Triggering receptor expressed on myeloid cells Like Transcript-1 (TLT-1) is exclusively expressed in platelets and promotes platelet aggregation. As the gene that encodes for TLT-1 is located in the TREM-1 gene cluster, this prompted us to investigate the expression of TREM-1 on platelets. Here we show that TREM-1 is constitutively expressed in α-granules and mobilised at the membrane upon platelet activation. Pharmacologic inhibition of TREM-1 reduces platelet activation as well as platelet aggregation induced by collagen, ADP, and thrombin in human platelets. Aggregation is similarly impaired in platelets from Trem-1−/− mice. In vivo, TREM-1 inhibition decreases thrombus formation in a carotid artery model of thrombosis and protects mice during pulmonary embolism without excessive bleeding. These findings suggest that TREM-1 inhibition could be useful adducts in antiplatelet therapies.Supplementary Material to this article is available online at www.thrombosis-online.com.

In vivo Effect of Insulin to Decrease Matrix Metalloproteinase-2 and -9 Activity after Arterial Injury

2013 ◽  
Vol 50 (4) ◽  
pp. 279-288 ◽  
Author(s):  
June Guo ◽  
Jiwanjeet K. Dhaliwall ◽  
Kalam K. Chan ◽  
Husam Ghanim ◽  
Nael Al Koudsi ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Arterial Injury ◽  
Matrix Metalloproteinase 2

EFFECTS OF SULFINPYRAZONE AND ITS METABOLITE G25671 ON PLATELET ACTIVATION AND DESENSITIZATION AND ON BRONCHOCONSTRICTION INDUCED BY THE PROSTAGLANDIN ENDOPEROXIDE ANALOGUE U46619

1987 ◽  
Author(s):  
M Hatmi ◽  
A Del Maschio ◽  
J Lefort ◽  
G De Gaetano ◽  
B B Varqaftiq ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activation ◽  
Ex Vivo ◽  
Oral Treatment ◽  
Human Platelets ◽  
Cyclooxygenase Inhibitors ◽  
Before And After ◽  
A Site

In previous studies we have found (Br. 3. Pharmac. 85, 849, 1985) that a) human platelets pre-exposed to arachidonic acid or to the endoperoxide analogue, U46619 and then washed and resuspended, fail to respond to a second challenge by both arachidonic acid and U46619; b) desensitization by arachidonic acid and U46619 occurs at a site sensitive to endoperoxides / thromboxane (Tx) receptor antagonists; c) the desensitizing effects of U46619 are direct, whereas those of arachidonic acid are mediated by a cyclooxygenase-dependent metabolite. Sulfinpyrazone (100 μM) and its thioether metabolite G25671 (50 μM) are known to suppress arachidonic acid-induced platelet aggregation and TxB2 formation (Eur. 3. Pharmac, 101, 209, 1984). We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevents desensitization. Platelet activation by the endoperoxide analogue U46619 is also prevented by sulfinpyrazone or G25671 (0.3-1 mM). The threshold aggregating concentrations of arachidonic acid and U46619 in healthy subjects before and after oral treatment with sulfinpyrazone were elevated by 2-3 fold and a good correlation between ex vivo and in vitro findings was established. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect.Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.

Abstract 489: Differential Phosphoproteomic Analysis of Platelet Activation by Specific Oxidized Phospholipids and Thrombin Reveals Mechanisms of Platelet Activation in Hyperlipidemia

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Alejandro Zimman ◽  
Bjoern Titz ◽  
Evangelia Komisopoulou ◽  
Thomas G Graeber ◽  
Eugene A Podrez
Keyword(s):  
Signaling Pathways ◽  
Signaling Pathway ◽  
Platelet Activation ◽  
Scavenger Receptor ◽  
Human Platelets ◽  
Oxidized Phospholipids ◽  
Systematic Analysis ◽  
Bioinformatic Tools ◽  
Induced Changes

We previously showed that specific oxidized phospholipids (oxPC CD36 ) activate platelets via the scavenger receptor CD36 and promote platelet hyper-reactivity in hyperlipidemia, however the signaling pathway(s) induced in platelets by oxPC CD36 are not defined. We employed mass spectrometry-based phosphoproteomics for the unbiased analysis of changes in protein phosphorylation induced by oxPC CD36 and thrombin, a strong platelet agonist, in human platelets. oxPC CD36 induced changes in phosphorylation of 148 unique phosphorylation sites (116 proteins) while thrombin induced changes of 297 unique sites (181 proteins). Most of the changes in phosphorylation induced by oxPC CD36 and thrombin identified in our study have never been reported before in platelets and include high- and low-abundant proteins with diverse molecular functions located in the plasma membrane, cytosol, or cytoskeleton. Analysis using multiple bioinformatic tools identified protein interaction networks, signaling pathways, activated kinases, and enriched phosphorylation motifs. Comparison between platelet agonists revealed multiple differences including the specific activation of a signaling pathway involving Src-family kinases (SFK), SYK kinase, and PLCγ2 by oxPC CD36 . Subsequent biochemical studies in human platelets demonstrated that this pathway is critical for platelet activation by oxPC CD36 and is downstream of CD36. In conclusion, systematic analysis of platelet activation pathways provided novel insights into the mechanism of platelet activation and specific signaling pathways induced by oxidized phospholipids that modulate platelet function in vivo in hyperlipidemia.

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.

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