Temporal Roles of Platelet and Coagulation Pathways in Collagen- and Tissue Factor-Induced Thrombus Formation

In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.

Nutrition phytochemicals affecting platelet signaling and responsiveness: implications for thrombosis and hemostasis

Cardiovascular disease, in particular due to arterial thrombosis, is a leading cause of mortality and morbidity, with crucial roles of platelets in thrombus formation. For multiple plant-derived phytochemicals found in common dietary components, claims have been made regarding cardiovascular health and antiplatelet activities. Here we present a systematic overview of the published effects of common phytochemicals, applied in vitro or in nutritional intervention studies, on agonist-induced platelet activation properties and platelet signaling pathways. Comparing the phytochemical effects per structural class, we included general phenols: curcuminoids (e.g., curcumin), lignans (honokiol, silybin), phenolic acids (caffeic and chlorogenic acid), derivatives of these (shikimic acid) and stilbenoids (isorhapontigenin, resveratrol). Furthermore, we evaluated the flavonoid polyphenols, including anthocyanidins (delphinidin, malvidin), flavan-3-ols (catechins), flavanones (hesperidin), flavones (apigenin, nobiletin), flavonols (kaempferol, myricetin, quercetin), isoflavones (daidzein, genistein); and terpenoids including carotenes and limonene; and finally miscellaneous compounds like betalains, indoles, organosulfides (diallyl trisulfide) and phytosterols. We furthermore discuss the implications for selected phytochemicals to interfere in thrombosis and hemostasis, indicating their possible clinical relevance. Lastly, we provide guidance on which compounds are of interest for further platelet-related research.

ANXA7 Regulates Platelet Lipid Metabolism and Ca 2+ Release in Arterial Thrombosis

Rationale: Platelet activation after contact to subendothelial collagen leads to acute arterial thrombosis. Annexin A7 (ANXA7) is a phospholipid-binding protein participating in the regulation of intracellular Ca 2+ and exocytosis. Objective: The present study aimed to determine the role of ANXA7 in platelet Ca 2+ signaling and lipid metabolism during platelet activation in arterial thrombosis using the ANXA7 inhibitor ABO and gene-targeted mice lacking Anxa7 ( Anxa7 -/- ). Methods and Results: ANXA7 is strongly expressed in platelets. Functionally, luminescence aggregometry revealed significantly abrogated aggregation and secretion of ABO-treated or Anxa7 -/- platelets when compared with untreated or Anxa7+/+ platelets after activation with collagen or the GPVI-specific agonist collagen-related peptide (CRP). Furthermore, while both thrombus formation on collagen-coated surfaces under high arterial shear rates in ABO-treated or Anxa7-deficient whole blood, and thrombotic vascular occlusion after FeCl3-induced injury in vivo in Anxa7 -/- bone marrow chimeric mice were significantly diminished, no prolongation of bleeding time was observed in ABO-treated or Anxa7 -/- mice. Fura-2-AM spectrofluorimetry unraveled a blunted [Ca2+]i increase in ABO-treated or Anxa7 -/- platelets after GPVI stimulation. Due to an abolished PLCy2 phosphorylation, Anxa7 -/- platelets displayed abrogated intracellular Ca 2+ mobilization following CRP-dependent platelet activation. Quantitative lipidomics analysis further revealed that ANXA7 critically affects platelet oxylipin metabolism following GPVI-dependent platelet activation. Anxa7 -/- platelets showed a significantly reduced generation of several bioactive metabolites, particularly TxA 2 and 12(S)-HETE. Finally, defective PLCy2 phosphorylation and blunted [Ca 2+ ]i increase in Anxa7 -/- platelets could be rescued by exogenous addition of 12(S)-HETE, indicating that ANXA7 is a critical regulator of the platelet 12-lipoxygenase in GPVI-dependent platelet Ca 2+ signaling during arterial thrombosis. Conclusions: The present study unravels ANXA7 as a regulator of oxylipin metabolism and Ca 2+ -dependent platelet activation downstream of GPVI. ANXA7 plays an important role in platelet signaling during arterial thrombosis and thus may reflect a promising target for novel antiplatelet strategies.

Exposure of Platelets to Dengue Virus and Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Platelet Cell Death and Thrombocytopenia in Mice

In tropical and subtropical regions, mosquito-borne dengue virus (DENV) infections can lead to severe dengue, also known as dengue hemorrhage fever, which causes bleeding, thrombocytopenia, and blood plasma leakage and increases mortality. Although DENV-induced platelet cell death was linked to disease severity, the role of responsible viral factors and the elicitation mechanism of abnormal platelet activation and cell death remain unclear. DENV and virion-surface envelope protein domain III (EIII), a cellular binding moiety of the virus particle, highly increase during the viremia stage. Our previous report suggested that exposure to such viremia EIII levels can lead to cell death of endothelial cells, neutrophils, and megakaryocytes. Here we found that both DENV and EIII could induce abnormal platelet activation and predominantly necrotic cell death pyroptosis. Blockages of EIII-induced platelet signaling using the competitive inhibitor chondroitin sulfate B or selective Nlrp3 inflammasome inhibitors OLT1177 and Z-WHED-FMK markedly ameliorated DENV- and EIII-induced thrombocytopenia, platelet activation, and cell death. These results suggest that EIII could be considered as a virulence factor of DENV, and that Nlrp3 inflammasome is a feasible target for developing therapeutic approaches against dengue-induced platelet defects.

132 A Novel Murine Model of Burn-Induced Platelet Dysfunction

Introduction Platelet dysfunction has been demonstrated as a part of burn induced coagulopathy (BIC), however, the etiology and clinical significance are unknown. Determining the etiology and clinical significance of BIC platelet dysfunction is difficult in humans due to heterogeneity of injuries and treatment. The goal of this study was to develop a murine model of BIC burn-induced platelet dysfunction to allow for high-throughput investigation of the mechanisms and the possible effects platelet dysfunction has on burn outcomes. Methods Using an established murine model of burn injury, we investigated plasma and cellular markers of BIC. Under adequate anesthesia and analgesia six-week-old C57BL6/J mice were administered a ~30% TBSA dorsal burn by scalding. Sham animals received identical preparation and resuscitation without the burn injury. Blood was collected at 6-, 24-, and 48-hours post-burn for measurement of platelet function, and plasma was isolated for protein measurements of coagulopathy (N=5 per group per time point). Results Like findings in humans, mice exhibited systemic markers of BIC (excess coagulation, fibrinolysis, and inflammation) within the first 6–24 hours post-burn. Platelets in whole blood were treated with platelet agonists ADP and PAR4-activating peptide (PAR4AP), and markers of platelet signaling and function (P-selectin expression and GpIIb/IIIa activation) were measured by flow cytometry. At 6 hours post-procedure, platelets from burn mice exhibited a slight, insignificant increase in markers of activation and response to stimulation compared with platelets from sham mice. At 24 hours post-burn, burn mice exhibited a significant decrease in platelet count (P< 0.02) and platelet function indicated by reduced GpIIb/IIIa activation (P< 0.01) and P-selectin expression (P< 0.05)in response to ADP and PAR4-AP compared with sham mice. Platelet function began to return at 48 hours post-burn with no significant difference between groups. Conclusions Platelet loss and dysfunction occur after burn injury, but the consequence of these effects is not well understood. The findings in this study are consistent across multiple experiments and resembled platelet dysfunction observed in different human traumatic injuries, validating the murine model as an inexpensive and efficient model of human injury in which to study platelet defects and the molecular mechanisms driving them.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region

Objective: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Conclusions: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.

Thromboxane A2 Deficit Diagnosis in a Patient with Suspicion of Von Willebrand Disease

Thromboxane A2 receptor deficiency is a qualitative platelet disorder that partially impedes adequate platelet signaling and aggregation. Generally, these patients have mild hemorrhagic manifestations in basal conditions, but may show severe bleeding when faced with a hemostatic challenge. We present the case of a 30-year-old female patient that arrives at the Hematology service with an undiagnosed bleeding disorder. She presented hemorrhagic complications during an augmentation mammoplasty and during an exodontia procedure, yet, during a C-section she presented none. At the first consultation she had normal coagulation factor levels by coagulometry, and normal platelet aggregation test for ADP, ristocetin, collagen and epinephrine. A platelet aggregation test for arachidonic acid confirmed thromboxane A2 deficit disorder. Thromboxane A2 deficit disorder is a rare qualitative platelet disorder that requires an extensive knowledge of coagulation and platelet function and tests, and a high level of clinical suspicion. These tests are especially difficult to interpret during pregnancy due to normal modifications to bodily function during this process.

Comparison of the central human and mouse platelet signaling cascade by systems biological analysis

Background Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC). Results The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12. Conclusions Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.