scholarly journals Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4883-4883
Author(s):  
Stephanie Renna ◽  
Leonard C. Edelstein ◽  
Steven Edward McKenzie
Keyword(s):  
Amino Acid ◽  
Platelet Activation ◽  
Knockout Mice ◽  
Transmembrane Domain ◽  
Conflicts Of Interest ◽  
Wild Type Mouse ◽  
Human Platelets ◽  
Amino Acid Sequences ◽  
Wild Type

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

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.

Biphasic Roles for the Soluble Guanylyl Cyclase (sGC) In Platelet Activation In Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 486-486
Author(s):  
Guoying Zhang ◽  
Binggang Xiang ◽  
Radek C. Skoda ◽  
Susan S. Smyth ◽  
Xiaoping Du ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Guanylyl Cyclase ◽  
Conflicts Of Interest ◽  
Soluble Guanylyl Cyclase ◽  
Wild Type ◽  
No Donor ◽  
Cgmp Production ◽  
Deficient Mice

Abstract Abstract 486 The role of intracellular secondary messenger cGMP in platelet activation has been controversial, with both stimulatory and inhibitory roles reported. The platelet cGMP is believed to be predominantly synthesized by soluble guanylyl cyclase (sGC), which is activated by nitric oxide (NO). To specifically determine the role of sGC-dependent cGMP synthesis in platelet function and in vivo thrombosis and hemostasis, we produced mice harboring a “floxed” sGC beta1 allele. In the “floxed” sGC beta1 mice (sGC beta1fl/fl), the exons 7 and 8 of sGC beta1 gene and an inserted Neo cassette were flanked with three LoxP sites. Platelet-specific deletion of sGC beta1fl/fl allele was accomplished through breeding of the sGC beta1fl/fl mice with pf4-Cre recombinase transgenic mice. Immunoblotting showed the complete absence of this protein in sGC beta1fl/fl/Cre platelets. Mice lacking sGC beta1 in platelets appeared to develop normally and had normal blood counts, including platelets. Blood pressure of platelet-specific sGC deficient mice was comparable to that of wild-type littermates. Inactivating the sGC beta1 gene in platelets abolished cGMP production induced by either NO donors or platelet agonists that are known to activate endogenous NO synthesis, confirming that both the platelet agonist-induced and NO donor-induced platelet cGMP production are predominantly mediated by sGC. Platelets lacking sGC exhibit a marked defect in aggregation and secretion in response to low doses of platelet agonists, collagen and thrombin. Importantly, tail-bleeding times were significantly prolonged in the platelet-specific sGC deficient mice compared with the wild-type littermates. In a FeCl3-induced carotid artery thrombosis model, time to occlusive thrombosis was prolonged in the platelet-specific sGC deficient mice, compared to wild type littermates. Thus, the agonist-stimulated sGC activation is important in promoting platelet granule secretion and aggregation. On the other hand, NO donor SNP-induced inhibition of platelet activation was abolished in sGC-deficient platelets. However, at high concentrations (>100μM), SNP inhibited platelet activation in both wild type and sGC deficient mice, indicating that both cGMP-dependent and -independent mechanisms are involved in NO donor-induced inhibition of platelet activation. Together, our data demonstrate that sGC contributes to both agonist-induced platelet activation and NO donor-induced platelet inhibition. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1131-1131
Author(s):  
Jasna Marjanovic ◽  
Brad Rumancik ◽  
Luke Weber ◽  
Felix Wangmang ◽  
Dane Fickes ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Glycogen Synthase ◽  
Thrombus Formation ◽  
Type I ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

Desialylation: A Novel Platelet Clearance Mechanism and a Potential New Therapeutic Target in Anti-GPIb Antibody Mediated Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 265-265 ◽  
Author(s):  
Dianne E. van Der Wal ◽  
Guangheng Zhu ◽  
June Li ◽  
Brian Vadasz ◽  
Yougbare Issaka ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Ivig Therapy ◽  
Reticuloendothelial System ◽  
Human Platelets ◽  
Cross Reactivity ◽  
Clearance Mechanism ◽  
Recent Retrospective Study

Abstract Abstract 265 Background: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibodies directed at patient's own platelet antigens, primarily glycoprotein (GP)IIbIIIa-integrin (70–80%) and GPIb-complex (20–40%). Current paradigm suggests that clearance of opsonized platelets through the reticuloendothelial system via Fcγ-receptors results in thrombocytopenia and bleeding disorders. However, evidence from others and our group demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP-models (Blood 2006). These observations are consistent with subsequent IVIG studies in human ITP patients. Interestingly, human anti-GPIb-mediated ITP patients seem also resistant to steroid therapy in our recent retrospective study (American Journal of Hematology 2012). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a different mechanism which is currently poorly understood. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa or GPIba deficient mice. Some of the mAbs have cross-reactivity to both mouse and human GPIIbIIIa and GPIba. Flow cytometry was used to evaluate whether these mAbs were able to induce platelet activation, apoptosis and desialylation. GPIbα is heavily glycosylated and the role of desialylation and exposure of underlying galactose and β-N-acetyl-D-glucosamine (βGN) residues on GPIbα in platelet clearance was assessed using the sialidase neuraminidase (NA) and it's inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). Desialyation effects on platelet activation and apoptosis was measured by flow cytometry. We also repeated these experiments with human platelets and plasma from human ITP-patients. We also investigated the effects of anti-GPIbα antibodies on platelet activation, apoptosis and clearance in vivo. Briefly, BALB/c mice were injected with anti-GPIbαor anti-GPIIIa mAbs and 24 hrs later, platelet desialylation, activation and apoptosis were measured by flow cytometry. The effect of desialylation on platelet clearance was assessed with DANA. The possible roles of Ashwell-Morell and MAC-1 receptors in GPIbα-mediated platelet clearance in the liver were examined using immunohistochemistry (anti-CD11b) or blocking of the Ashwell-Morell receptor with asialofetuin. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa mAbs, induced significant P-selectin expression and phosphatidylserine (PS)-exposure, and increased inner membrane mitochondrial depolarization (ΔYm). Interestingly, platelets were desialylated in the presence of anti-GPIbα but not anti-GPIIbIIIa mAbs. Moreover, we found that desialylation of GPIbα lies directly upstream of platelet activation and apoptosis, as prior treatment with DANA diminished PS-exposure, and P-selectin expression. Most importantly, incubations of human platelets with ITP-patient plasma showed similar effects. In vivo, we found significant increases in PS-exposure and ΔYm induced by anti-GPIbα, but not by anti-GPIIIa mAbs, independent of IgG subclass. Interestingly, prior injection with DANA rescued platelets numbers in anti-GPIbα, but not in anti-GPIIIa injected mice. A significant role for the Ashwell-Morell and MAC-1 receptors in the clearance of deglycosylated platelets was observed; blocking of the Ashwell-Morell receptors by asialofetuin, decreased platelet clearance in anti-GPIbα, but not anti-GPIIbIIIa antibody injected mice, there was also increased staining for MAC-1 on Kupffer cells, exclusively in the presence of an anti-GPIbα mAb tested. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce GPIbα desialyation, leading to platelet activation and apoptosis. Therefore, we identified novel Fc-independent platelet clearance pathways, more specifically, via Ashwell-Morell and MAC-1 receptors on hepatocytes and liver macrophages. These findings may lead to novel therapeutic regimens including the potential use of sialidase inhibitors as a solution for anti-GPIb-mediated ITP patients previously refractory to both steroid and IVIG therapies. Disclosures: No relevant conflicts of interest to declare.

Platelet Desialylation: A Novel Mechanism of Fc-Independent Platelet Clearance and a Potential Diagnostic Biomarker and Therapeutic Target in immune Thrombocytopenia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 467-467
Author(s):  
June Li ◽  
Dianne Evertdina van der Wal ◽  
Guangheng Zhu ◽  
Miao Xu ◽  
Issaka Yougbare ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Ivig Therapy ◽  
Human Platelets ◽  
Diagnostic Biomarker ◽  
Large Patient ◽  
Sialidase Inhibitor

Abstract Background:Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa (70-80%) and/or GPIb-complex (20-40%). Current theory suggests antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc-FcγR interactions. However, evidence from us and others demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP models (Blood 2006) and subsequent IVIG studies in human ITP patients, including our recent large patient cohort study (JTH 2014). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a presently unidentified mechanism different than that of anti-GPIIbIIIa. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa-/- or GPIba-/- mice, which also recognize GPIbα and GPIIbIIIa of different species including human. Flow cytometry, immunofluorescence, and western blotting were used to evaluate whether these mAbs induced platelet activation, neuraminidase-1 translocation and desialylation of the heavily glycosylated GPIbα in the presence of sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). These experiments were repeated with human platelets and human ITP patient plasma. We further investigated the effects of anti-GPIbα antibodies on platelet activation, desialylation and clearance in vivo; BALB/c mice were injected with anti-GPIbα or anti-GPIIbIIIa mAbs and following, platelet activation and desialylation were measured by flow cytometry. Hepatocytic Ashwell-Morell receptor (AMR) mediated anti-GPIbα platelet clearance in the liver was examined using immunohistochemistry or blocking the AMR with asialofetuin in both wild-type and macrophage depleted mice. Therapeutic administration of DANA in a murine ITP model assessed the significance of Fc-independent anti-GPIbα mediated platelet clearance in ITP. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induced significant P-selectin expression, JON/A binding, neuraminidase-1 translocation and desialylation in murine platelets. Interestingly, certain human platelets were activated (P-selectin expression) and desialylated in the presence of both anti-GPIbα and anti-GPIIbIIIa mAbs or ITP patient plasma. However, we demonstrate that the anti-GPIIbIIIa antibody mediated platelet effects are dependent on the FcγRIIa present exclusively on human platelets as FcγRII blocker IV.3 completely attenuated the response. In contrast, IV.3 had little effect on anti-GPIbα mediated platelet activation or desialylation. Anti-GPIbα Fab fragments and platelet signal pathway inhibitors demonstrate that anti-GPIbα mediated platelet activation and desialylation are consequences of GPIbα cross linking and are reinforced by a positive feedback loop. In vivo, we found significant increases in P-selectin and desialylation in anti-GPIbα injected mice, independent of IgG subclass. A significant role for the hepatic AMR in the clearance of deglycosylated platelets was observed; particularly in macrophage depleted mice whereby, although anti-GPIIbIIIa mediated platelet clearance was completely attenuated, anti-GPIbα mediated platelets clearance still occurred, but was completely rescued with asialofetuin. Immunohistochemistry revealed significant co-localization of anti-GPIbα opsonized platelets with AMR. These suggest the AMR is the dominant Fc-independent anti-GPIbα mediated platelet clearance pathway in the absence of macrophages. Remarkably, sialidase inhibitor DANA ameliorated anti-GPIbα mediated thrombocytopenia in mice. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce platelet activation leading to GPIbα desialyation and platelet clearance via a novel Fc-independent pathway: the hepatic AMR. Our data also suggested that some anti-GPIIbIIIa autoantibodies in human patients may also induce platelet activation and desialylation via the platelet FcR signaling pathway. These findings may lead to novel therapeutic regimens including designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of both anti-GPIIbIIIa and anti-GPIbα mediated and/or refractory ITP. Disclosures No relevant conflicts of interest to declare.

HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi

2015 ◽  
Vol 114 (11) ◽  
pp. 994-1003 ◽  
Author(s):  
Michael Bode ◽  
David Haenel ◽  
Christoph E. Hagemeyer ◽  
Hannah Seeba ◽  
Daniel Duerschmied ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Platelet Activation ◽  
Knockout Mice ◽  
Surface Expression ◽  
Human Platelets ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Specific Expression ◽  
Platelet Surface ◽  
Activated Platelets

SummaryHigh mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

Formation of Procoagulant Platelets in Heparin-Induced Thrombocytopenia (HIT) Follows a Unique Signaling Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 197-197
Author(s):  
Pierrette Andre ◽  
Moritz Stolla ◽  
Donna Woulfe ◽  
Massiel Chavez ◽  
Wolfgang Bergmeier ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Wild Type Mouse ◽  
Annexin V ◽  
Human Platelets ◽  
Therapeutic Interventions ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Drug Induced ◽  
Platelet Factor ◽  
Considerable Morbidity

Abstract Abstract 197 HIT is a drug-induced, immune-mediated thrombocytopenia and thrombosis disorder associated with considerable morbidity and mortality. HIT is associated with generation of procoagulant platelet microparticles (Warkentin et al., Blood 1994; 84:3691), one of the cardinal features of procoagulant platelets, along with a phosphatidylserine-positive (PS+) external plasma membrane and surface retention of procoagulant proteins from plasma or released from platelet γ-granules. Other names for procoagulant platelets include COAT, or coated, platelets (Dale, J Thromb Haemostas 2005; 3:2185). To date, virtually all of the work on procoagulant platelets has used collagen or convulxin signaling via ITAM-associated GPVI, combined with thrombin signaling via GPCRs PAR1/PAR4. Batar and Dale reported the formation of coated platelets as a result of dual platelet FcγRIIa and thrombin stimulation; they used crosslinking of an anti-FcγRIIa antibody or anti-platelet glycoprotein antibodies which also engaged FcγRIIa (J Lab Clin Med 2001; 138:393). We report here the procoagulant activation of human and FcγRIIa transgenic (tg) mouse platelets stimulated by the HIT immune complex (IC) and thrombin (thr). HIT IC consist of ultralarge complexes of heparin and platelet factor 4 (hep/PF4) bound by HIT-like monoclonal antibody KKO. Washed human platelets, wild-type mouse platelets, or FcγRIIa tg mouse platelets were stimulated with the HIT IC + thr. Controls include convulxin (100 ng/ml) + thr, HIT IC alone, or thr alone as the stimulus. Human platelets and FcγRIIa-tg platelets, but not wild-type mouse platelets, develop a 40 to 60% PS+ population in response to HIT IC (100 ug/ml KKO)+ thr (0.5 U/ml), but not to either alone, as assessed by flow cytometry using labeled Annexin V. Unlike cvx + thr, which consistently shows a broad distribution of PS+ platelets in our hands and in the literature, HIT IC-stimulated platelets reproducibly show one discrete PS+ subpopulation (n = 6 per group). Preincubation of platelets with novel specific Syk inhibitor PRT318 (Reilly et al., Blood 2011; 117:2241) completely prevented PS+ platelet formation. FcγRIIa-tg platelets null for Akt2 showed a ∼50% reduction in the formation of PS+ platelets. Studies with FcγRIIa-tg mice null for Cal-DAG GEFI are ongoing. Understanding the unique pattern of procoagulant platelets generated by HIT IC + thr and the signals which produce it may lead to new diagnostic tests and therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Fucoidan Is a Novel Platelet Agonist for CLEC-2 Receptor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 94-94
Author(s):  
Bhanukanth Manne ◽  
Todd M Getz ◽  
Craig Hughes ◽  
Carol T Dangelmaier ◽  
Steve P Watson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Animal Models ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Lag Phase ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner

Abstract Abstract 94 Fucoidan, a sulphated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor (TFPI) (Prasad et al., Blood 111:672, 2008). The decrease in bleeding times in the hemophilia animal models by in vivo administration of fucoidan suggests the beneficial effect of fucoidan as a novel treatment. Furthermore, in vitro studies using platelet poor plasma from hemophilia animal models and human patients has shown that fucoidan inhibits TFPI thereby contributing to an increase in the extrinsic coagulation pathway activity. The effect of fucoidan on platelets however has not been studied. As it is known that the platelet count remains unaffected in hemophilia A patients and bleeding times are primarily measured to assess normal platelet function, we hypothesize that the decrease in bleeding times in the hemophilia animal models may be due to platelet activation by fucoidan. In this study, we demonstrate for the first time that fucoidan induces platelet activation in a concentration dependent manner. Fucoidan-induced platelet activation is completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, and in the absence of Syk and PLC-g2. Furthermore, fucoidan-induced platelet activation has a lag phase, which is reminiscent of platelet activation by collagen and by CLEC-2 receptor agonists. Platelet activation by fucoidan however was only slightly inhibited in FcRg-chain null mice indicating that fucoidan is not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in CLEC-2 deficient mouse platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data shows fucoidan as a novel CLEC-2 receptor agonist that activates platelets through an SFK-dependent signaling pathway. Further, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. . / B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. . / C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. Disclosures: No relevant conflicts of interest to declare.

Racial Differences In Thrombin-Induced Human Platelet PAR4 Reactivity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1054-1054
Author(s):  
Leonard C. Edelstein ◽  
Lukas M Simon ◽  
Raul Teruel Montoya ◽  
Michael Holinstat ◽  
Edward Chen ◽  
...  
Keyword(s):  
Racial Differences ◽  
Platelet Activation ◽  
Platelet Function ◽  
Racial Difference ◽  
Conflicts Of Interest ◽  
Platelet Reactivity ◽  
Wild Type Mouse ◽  
Induced Aggregation ◽  
Megakaryocytic Cell

Abstract Compared to white patients, black patients have worse outcomes after acute coronary events, but there is a paucity of literature considering racial differences in platelet function. Thrombin is an especially potent in vivo platelet agonist, and no work has considered racial differences in thrombin-induced platelet aggregation. Using PAR1- and PAR4- activation peptides (APs), we recently reported that platelets from healthy black subjects (n = 70) demonstrated greater aggregation to the PAR4-AP than platelets from white subjects (n = 84) (p = 5.15 x 10-8). There was no racial difference to PAR1-AP, ADP or CRP. The goal of the current study was to determine if this racial difference in PAR4-AP-mediated platelet reactivity was also observed with thrombin and to investigate responsible molecular and genetic mechanism(s). A detailed dose-response study (4 blacks and 3 whites) revealed that when thrombin signaling was restricted to PAR4 by inhibiting PAR1 with BMS-200261, platelets from black subjects aggregated faster than platelets from white subjects at low concentrations of thrombin. No PAR1-AP-induced aggregation occurred in the presence of BMS-200261. A subsequent replication study (an additional 5 blacks and 5 whites) again showed platelets from black subjects aggregated faster than white subjects in the absence of PAR1 signaling (p = 3.56x10-5). DNA from all 154 subjects was genotyped for 5 million SNPs (HumanOmni5 array) and principal component analysis revealed that the genotypes segregated into two distinct groups that correlated perfectly with subject self-identified race. Gene expression profiling on leukocyte-depleted platelets from all 154 subjects revealed numerous differentially expressed (DE) RNAs associated with both race and PAR4 reactivity. The gene encoding phosphatidylcholine transfer protein (PC-TP), PCTP, showed the strongest correlation with race (p = 10-23; q = 10-20) and with PAR4 reactivity (p = 3.4x10-8; q = 3.5x10-4). PC-TP protein was higher in platelets from blacks (p = 3.8x10-6) and levels correlated with reactivity to PAR4-AP (r = 0.249, p = 0.002). Pctp has been knocked out in mice, but we found that wild type mouse platelets express little or no Pctp protein, consistent with mouse platelet RNA data from Rowley et al (Blood 2011). However, a specific PC-TP inhibitor resulted in a reduced aggregation response to PAR4-AP, but not PAR1-AP. Transfection of a siRNA against PCTP reduced both PCTP mRNA and PC-TP protein levels, and inhibited Ca+ release in a megakaryocytic cell line, Meg-01 in response to PAR4-AP but not PAR1-AP. A racial difference in platelet Ca+ release in response to PAR4-AP treatment was also observed (p = 0.02). Platelet microRNA (miRNA) profiling from all 154 subjects also revealed numerous DE miRNAs associated with both race and PAR4 reactivity. Target prediction analysis indicated that miR-376c is a candidate for regulating PCTP expression. qRT-PCR of all 154 subjects indicated that miR-376c levels are expressed higher in platelets from whites (p = 1.47 x10-4; q = 1.38 x10-3), and are inversely correlated with PCTP mRNA (r = -0.214; p = 0.008), PC-TP protein (r = -0.211; p = 0.009) and PAR4 reactivity (r = -0.161; p = 0.049). Transfection of megakaryocytic cell lines or cord blood CD34+ derived megakaryocytes with the pre-miR-376c precursor or LNA-miR-376c inhibitor resulted in decreased (P<0.01) or increased PCTP (p = 0.0003), respectively. Co-transfection of the miRNA precursor or inhibitor with a luciferase vector containing the PCTP 3’UTR indicated the regulation was dependent on the predicted miR-376c target site. In summary, we have uncovered a racial difference in thrombin-induced PAR4 platelet activation. This finding has potential clinical significance because PAR4 is the primary means by which thrombin activates platelets in the presence of vorapaxar (a PAR1 inhibitor in clinical trials), and the risks and benefits of vorapaxar by race are unknown. This racial difference in platelet activation is mediated, in part, by PC-TP, a novel protein in platelet biology. Our data also supports racial differences in miRNA expression, one of which (miR-376c) regulates PC-TP expression. These results indicate a genomic contribution to platelet function that differs by race, emphasize a need to consider race effects when developing anti-thrombotic drugs and raise the possibility that PC-TP inhibition might be a useful anti-thrombotic strategy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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