Abstract 463: TLT-1 Deficiency Affects Hypercholesterolemia and Progression of the Atherosclerotic Plaque in Apoe Null Mice

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Marieli Gonzalez ◽  
Fiorella Reyes ◽  
Deborah Marrero ◽  
A V Washington
Keyword(s):  
Platelet Activation ◽  
Inflammatory Responses ◽  
Plaque Rupture ◽  
Atherosclerotic Lesion ◽  
Soluble Form ◽  
Chow Diet ◽  
Fatty Streak ◽  
Wild Type ◽  
Platelet Surface ◽  
Cholesterol Levels

Platelet activation at sites of inflammation triggers the secretion of molecules that induce the transition of atherosclerosis from fatty streak to an acute disease, featuring an increased vulnerability of the atherosclerotic lesion that results in plaque rupture and thrombosis. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a molecule exclusively found in the α-granules of megakarocytes and platelets and has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface, while its soluble form, s-TLT-1, is secreted and detected in serum. Studies using the C57Bl/6 treml1 - /- mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Because we have found that sTLT-1 levels are significantly elevated in apoE mice when compared to wild type, we hypothesized that TLT-1 may be playing an important role in the progression of atherosclerosis. To address this possibility, we generated apoE - /- / treml1 - /- double knockout mice [DN]. Assessment of lesions after 4 weeks high-fat diet (HFD) demonstrated that at early stages, TLT-1 deficiency accelerates fatty streak formation. After 20 weeks on HFD, lesions in both apoE - /- and [DN] mice progressed to an advance fibrous plaque stage. Although their lesion sizes were not substantially different, lesion compositions were. The mechanistic basis of these differences appears to be that the [DN] mice have significantly higher cholesterol levels when compared to apoE - /- mice. The increased cholesterol levels extend to the treml1 -/- mouse when compared to wild type mice at 4 weeks on HFD, this difference, however, gradually subsides as wild type mice cholesterol levels increase over 20 weeks. Interestingly, cholesterol levels in 50 week old mice on chow diet revealed minimal differences between test and control mice suggesting the higher cholesterol levels are related to increased dietary intake. Our work defines a surprising role for TLT-1 in the regulation of serum cholesterol levels during atherogenesis.

Abstract 156: Macrophage Low-Density Receptor--Related Protein 1 Deficiency Facilitates Atherosclerosis Regression in Hyperlipidemic Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Lin Zhu ◽  
Patricia G Yancey ◽  
Lei Ding ◽  
John L Blakemore ◽  
Youmin Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Atherosclerotic Lesion ◽  
Lesion Size ◽  
Chow Diet ◽  
Low Density ◽  
Related Protein ◽  
Wild Type ◽  
Lesion Area ◽  
Cholesterol Levels ◽  
Inflammatory Macrophages

Atherosclerosis regression is characterized by egress of macrophages out of the artery wall. We have previously shown that macrophages lacking low-density receptor related protein 1 (MFLRP1-/-) are pro-inflammatory and lead to increased lesion formation in apoE-/- mice. To study the role of macrophage inflammation during atherosclerosis regression, bone marrow from four different types of mice (wild-type, MFLRP1-/-, apoE-/- and apoE-/-/ MFLRP1-/-) was transplanted into apoE-/- recipient mice who had been fed a Western-type diet for 12 weeks. ApoE-/- recipient mice transplanted with apoE-/- bone marrow were sacrificed 2 weeks post-BMT for determination of baseline aortic atherosclerosis. After 8 weeks on chow diet, cholesterol levels were normalized in mice reconstituted with wild-type (WT) and MFLRP1-/- bone marrow (157± 36 mg/dl and 136 ± 33 mg/dl, respectively), and significantly lowered in mice with apoE-/- (302±33 mg/dl) or apoE-/-/ MFLRP1-/- (294±52 mg/dl) macrophages compared to baseline (387±34 mg/dl). Total atherosclerotic lesion area in the aortic root decreased by 15% in mice receiving WT macrophages, and decreased by an additional 10% in mice transplanted with LRP1 deficient macrophages (p<0.05 compared to WT). Similarly, mice reconstituted with apoE-/-/ MFLRP1-/- bone marrow had 15% (p < 0.01) smaller lesion size than mice receiving apoE-/- marrow. The lesion area positive for CD68 was significantly smaller in MFLRP1-/- mice compared to WT mice, and in apoE-/-/ MFLRP1-/- mice compared to apoE-/- mice. The ratio of necrotic to total lesion area was significantly lowered by WT and LRP1-/- macrophages, and was also reduced in recipients of apoE-/-/MFLRP1-/- compared to apoE-/- bone marrow. Here we demonstrate that absence of LRP1 in macrophages, which is known to cause pro-inflammatory changes, promotes atherosclerosis regression. Our study supports the novel idea that pro-inflammatory macrophages efficiently egress from the plaque in a regressive environment caused by switching from a high-fat to a chow diet. This observation sets the stage for a change in paradigm on how to target inflammation for prevention of atherosclerotic cardiovascular events.

Spontaneous Atherosclerotic Lesions in TREM-Like Transcript-1 Deficient Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3312-3312
Author(s):  
Marieli Gonzalez ◽  
Fiorella Reyes ◽  
Deborah Marrero ◽  
A. Valance Washington
Keyword(s):  
Vessel Wall ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Atherogenic Diet ◽  
Chow Diet ◽  
Aortic Sinus ◽  
Platelet Surface ◽  
Atherosclerosis Progression ◽  
Atherosclerotic Lesions ◽  
Cholesterol Levels

Abstract Abstract 3312 It is well known that platelets, aside from regulating hemostasis, play an important role in inflammation-associated conditions like sepsis, viral infections, and atherosclerosis. In the latter, platelets not only form occlusive thrombi at lesions, but also play a role in the initiation of the disease by depositing activating molecules such as cytokines on the developing plaque. Although the mechanism by which platelet aggregation leads to occlusion is well-defined, the role of platelets in lesion initiation and progression is poorly understood and thus remains a gap in our knowledge. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a receptor exclusively found on megakarocytes and platelets that has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface along with p-selectin from the α-granules. Studies using the treml1−/− mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms and therefore should play a role in the development of atherosclerosis. Our original hypothesis was that evaluation of atherosclerosis lesions in the treml1−/− mouse would demonstrate fewer lesions and hence, a similar phenotype as the psel−/− mouse. Evaluation of 50 week old mice fed a chow diet surprisingly revealed spontaneous lesions in C57Bl/6 treml1−/− mice. Subsequent evaluation of cholesterol levels in treml1−/−mice on an atherogenic diet for four weeks demonstrated that they have significantly higher cholesterol levels when compared to WT mice. To evaluate atherosclerosis progression in TLT-1 deficiency, we developed the apoE−/−/treml1−/− double knockout mice and assessed lesion development after a four weeks atherogenic diet. Our results demonstrate that double null mice have exacerbated lesions when compared to the apoE −/− mice. Accordingly, 50 week old double null mice fed a chow diet also have larger atherosclerotic lesions in the aortic sinus than apoE −/− mice. These results clearly support a role for TLT-1 in dampening the progression of atherosclerosis. Current data from our laboratory suggest that TLT-1 may affect leukocyte function, therefore based on our model that TLT-1 deficiency leads to a heighted inflammatory state we hypothesized that treml1−/− mice will demonstrate greater leukocyte recruitment into the injured vessel wall, leading to greater deposition of factors that recruit more platelets and macrophages that later become foam cells. To delineate the mechanism by which TLT-1 affects atherosclerosis progression, we have evaluated neutrophil and monocyte infiltration into the vessel wall of the aortic sinus after four weeks atherogenic diet. The current state of our investigation is reported here. Disclosures: No relevant conflicts of interest to declare.

Abstract 104: Apolipoprotein D: Implications for Multiple Roles in Lipid Metabolism

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Ujala Srivastava ◽  
Ehab M Abo-Ali ◽  
Susanna Nguy ◽  
Jean Lebegue ◽  
Kamilah Ali
Keyword(s):  
Lipid Metabolism ◽  
Plasma Cholesterol ◽  
Atherosclerotic Lesion ◽  
Minor Component ◽  
Chow Diet ◽  
Lcat Activity ◽  
Wild Type ◽  
Cholesterol Acyl Transferase ◽  
Cholesterol Levels ◽  
Significant Difference

Introduction- ApoD is a ubiquitously expressed protein that binds small hydrophobic ligands and is a minor component of lipoproteins. Polymorphisms of the human ApoD gene are associated with lipid abnormalities, specifically the reduction of HDL and ApoA1 levels. In fact, hepatic overexpression of ApoD has been shown to regulate the amount of plasma triglycerides. ApoD is also upregulated in human and mouse models of atherosclerosis, and is localized in cell types involved in atherosclerotic lesion formation. These data suggest that ApoD plays a role in lipid metabolism by modulating cellular processes in vascular cells during atherogenesis. In this study, our objective is to identify the role(s) of ApoD in lipid metabolism and to elucidate the mechanisms involved in this process. Methods and Results- To accomplish our objective, we used a two-pronged approach. We first studied the effect of ApoD on lipid metabolism on a chow diet. There was no significant difference between the levels of plasma cholesterol in ApoD -/- and wild-type mice on a chow diet; however, hepatic cholesterol levels had more than doubled. A 96-gene PCR array was used to assess differential expression of genes involved in fatty liver biogenesis. There was at least a 2-fold difference in expression in about 10 genes involved in insulin/glucose signaling, lipogenesis, and inflammation in the ApoD -/- mice. We then studied the effect of a Western diet in the ApoD -/- mice, which showed a significant reduction in plasma LDL-cholesterol and HDL-cholesterol when compared to wild-type mice. Analysis of the HDL fractions after subjecting plasma to a Fast Protein Liquid Chromatography column revealed increased levels of ApoA1 and Lecithin Cholesterol Acyl Transferase (LCAT) activity in ApoD -/- mice. Conclusion- A decrease in plasma cholesterol and an increase in ApoA1 and LCAT activity suggest that ApoD may play a role in the catabolism of HDL particles, resulting in lower plasma cholesterol levels in ApoD -/- mice. Our current data implies that ApoD plays a multifunctional role in lipid metabolism and the mechanism by which this occurs must be further examined.

Platelet P-selectin facilitates atherosclerotic lesion development

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2661-2666 ◽  
Author(s):  
Peter C. Burger ◽  
Denisa D. Wagner
Keyword(s):  
Atherosclerotic Lesion ◽  
Soluble Form ◽  
Wild Type ◽  
Lesion Development ◽  
Atherosclerotic Lesions ◽  
Lesion Growth ◽  
Microparticle Formation ◽  
Apoe Deficient Mouse ◽  
Atherosclerotic Lesion Development

P-selectin is an adhesion molecule expressed on activated platelets and endothelium. It is known to play an important role in atherosclerosis. P-selectin also circulates in plasma in a soluble form (sP-selectin), which induces procoagulant microparticle formation. We investigated the role of platelet versus endothelial P-selectin in generating sP-selectin and in the formation of atherosclerotic lesions in the apolipoprotein E (apoE)–deficient mouse model. For this we transplanted apoE−/−P-selectin−/− and apoE−/−P-selectin+/+ lethally irradiated mice with bone marrow of either genotype. Seven months after transplantation, we determined from the chimeric animals that the majority of circulating sP-selectin was of endothelial origin. Thus, in atherosclerosis, the procoagulant sP-selectin reflects endothelial rather than platelet activation. We found that endothelial P-selectin was crucial for the promotion of atherosclerotic lesion growth because in its absence only relatively small lesions developed. However, platelet P-selectin also contributed to the lesion development because lesions in wild-type recipients receiving transplants with wild-type platelets were 30% larger than those receiving P-selectin-deficient platelets (P < .008) and were more frequently calcified (80% versus 44%). In comparison with P-selectin wild-type animals, absence of either endothelial or platelet P-selectin inhibited migration of smooth muscle cells into the lesion. Thus, in addition to endothelium, platelets and their P-selectin also actively promote advanced atherosclerotic lesion development.

scholarly journals Roles of protease-activated receptors in a mouse model of endotoxemia

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3912-3921 ◽  
Author(s):  
Eric Camerer ◽  
Ivo Cornelissen ◽  
Hiroshi Kataoka ◽  
Daniel N. Duong ◽  
Yao-Wu Zheng ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Disseminated Intravascular Coagulation ◽  
Inflammatory Responses ◽  
Endothelial Activation ◽  
Wild Type ◽  
Protease Activated Receptors ◽  
Endotoxin Challenge ◽  
Intravascular Coagulation ◽  
Endotoxemia Model ◽  
Independent Process

Endotoxemia is often associated with extreme inflammatory responses and disseminated intravascular coagulation. Protease-activated receptors (PARs) mediate cellular responses to coagulation proteases, including platelet activation and endothelial cell reactions predicted to promote inflammation. These observations suggested that PAR activation by coagulation proteases generated in the setting of endotoxemia might promote platelet activation, leukocyte-mediated endothelial injury, tissue damage, and death. Toward testing these hypotheses, we examined the effect of PAR deficiencies that ablate platelet and endothelial activation by coagulation proteases in a mouse endotoxemia model. Although coagulation was activated as measured by thrombin-antithrombin (TAT) production and antithrombin III (ATIII) depletion, Par1–/–, Par2–/–, Par4–/–, Par2–/–:Par4–/–, and Par1–/–:Par2–/– mice all failed to show improved survival or decreased cytokine responses after endotoxin challenge compared with wild type. Thus, our results fail to support a necessary role for PARs in linking coagulation to inflammation or death in this model. Interestingly, endotoxin-induced thrombocytopenia was not diminished in Par4–/– mice. Thus, a mechanism independent of platelet activation by thrombin was sufficient to cause thrombocytopenia in our model. These results raise the possibility that decreases in platelet count in the setting of sepsis may not be caused by disseminated intravascular coagulation but instead report on a sometimes parallel but independent process.

P-Selectin and Platelet Clearance

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4446-4452 ◽  
Author(s):  
Gaëtan Berger ◽  
Daqing W. Hartwell ◽  
Denisa D. Wagner
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Activation ◽  
Soluble Form ◽  
Wild Type ◽  
Adhesion Receptor ◽  
Activated Platelets ◽  
Deficient Mice

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

scholarly journals TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1050-1050
Author(s):  
Angela Doerr ◽  
Denise Pedrosa ◽  
Maria Schander ◽  
Yotis A. Senis ◽  
Alexandra Mazharian ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Type I Collagen ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Type I ◽  
Wild Type ◽  
Platelet Surface ◽  
Knock Out ◽  
Integrin Αiibβ3

Abstract Background Thrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma. Results VWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1. Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice. Conclusions This study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

Maternal high-fat feeding in pregnancy programs atherosclerotic lesion size in the ApoE*3 Leiden mouse

2016 ◽  
Vol 7 (3) ◽  
pp. 290-297 ◽  
Author(s):  
E. J. Tarling ◽  
K. J. P. Ryan ◽  
R. Austin ◽  
S. J. Kugler ◽  
A. M. Salter ◽  
...  
Keyword(s):  
Plasma Cholesterol ◽  
Maternal Diet ◽  
Atherosclerotic Lesion ◽  
Plasma Lipid ◽  
Lesion Size ◽  
Atherogenic Diet ◽  
Chow Diet ◽  
Wild Type ◽  
High Fat ◽  
Cell Proliferation And Differentiation

Periods of rapid growth seen during the early stages of fetal development, including cell proliferation and differentiation, are greatly influenced by the maternal environment. We demonstrate here that over-nutrition, specifically exposure to a high-fat dietin utero, programed the extent of atherosclerosis in the offspring of ApoE*3 Leiden transgenic mice. Pregnant ApoE*3 Leiden mice were fed either a control chow diet (2.8% fat,n=12) or a high-fat, moderate-cholesterol diet (MHF, 19.4% fat,n=12). Dams were fed the chow diet during the suckling period. At 28 days postnatal age wild type and ApoE*3 Leiden offspring from chow or MHF-fed mothers were fed either a control chow diet (n=37) or a diet rich in cocoa butter (15%) and cholesterol (0.25%), for 14 weeks to induce atherosclerosis (n=36). Offspring from MHF-fed mothers had 1.9-fold larger atherosclerotic lesions (P<0.001). There was no direct effect of prenatal diet on plasma triglycerides or cholesterol; however, transgenic ApoE*3 Leiden offspring displayed raised cholesterol when on an atherogenic diet compared with wild-type controls (P=0.031). Lesion size was correlated with plasma lipid parameters after adjustment for genotype, maternal diet and postnatal diet (R2=0.563,P<0.001). ApoE*3 Leiden mothers fed a MHF diet developed hypercholesterolemia (plasma cholesterol two-fold higher than in chow-fed mothers,P=0.011). The data strongly suggest that maternal hypercholesterolemia programs later susceptibility to atherosclerosis. This is consistent with previous observations in humans and animal models.

P-Selectin and Platelet Clearance

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4446-4452 ◽  
Author(s):  
Gaëtan Berger ◽  
Daqing W. Hartwell ◽  
Denisa D. Wagner
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Activation ◽  
Soluble Form ◽  
Wild Type ◽  
Adhesion Receptor ◽  
Activated Platelets ◽  
Deficient Mice

Abstract P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

scholarly journals Dapagliflozin reduces thrombin generation and platelet activation: implications for cardiovascular risk reduction in type 2 diabetes mellitus

Diabetologia ◽  
2021 ◽  
Author(s):  
Christina Kohlmorgen ◽  
Stephen Gerfer ◽  
Kathrin Feldmann ◽  
Sören Twarock ◽  
Sonja Hartwig ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Cardiovascular Risk ◽  
Blood Glucose ◽  
Platelet Activation ◽  
Thrombin Generation ◽  
Atherosclerotic Lesion ◽  
Hdl Cholesterol ◽  
Cholesterol Levels ◽  
The Impact

Abstract Aims/hypothesis People with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium–glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis. Methods Male, 8-week-old LDL-receptor-deficient (Ldlr−/−) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks. Results Dapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet–leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr−/− mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10−9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10−9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis. Conclusions/interpretation We demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin–platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin’s beneficial cardiovascular risk profile. Graphical abstract

Sign in / Sign up

Export Citation Format

Share Document