Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbß3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbß3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

Pediatric Swine Model of Methicillin-Resistant Staphylococcus aureus Sepsis-Induced Coagulopathy, Disseminated Microvascular Thrombosis, and Organ Injuries.

Sepsis-induced coagulopathy leading to disseminated microvascular thrombosis is associated with high mortality and has no existing therapy. Despite the high prevalence of Gram-positive bacterial sepsis, especially methicillin-resistant Staphylococcus aureus (MRSA), there is a paucity of published Gram-positive pediatric sepsis models. Large animal models replicating sepsis-induced coagulopathy are needed to test new therapeutics before human clinical trials. HYPOTHESIS: Our objective is to develop a pediatric sepsis-induced coagulopathy swine model that last 70 hours. METHODS AND MODELS: Ten 3 weeks old piglets, implanted with telemetry devices for continuous hemodynamic monitoring, were IV injected with MRSA (n = 6) (USA300, Texas Children's Hospital 1516 strain) at 1 × 109 colony forming units/kg or saline (n = 4). Fluid resuscitation was given for heart rate greater than 50% or mean arterial blood pressure less than 30% from baseline. Acetaminophen and dextrose were provided as indicated. Point-of-care complete blood count, prothrombin time (PT), activated thromboplastin time, d-dimer, fibrinogen, and specialized coagulation assays were performed at pre- and post-injection, at 0, 24, 48, 60, and 70 hours. Piglets were euthanized and necropsies performed. RESULTS: Compared with the saline treated piglets (control), the septic piglets within 24 hours had significantly lower neurologic and respiratory scores. Over time, PT, d-dimer, and fibrinogen increased, while platelet counts and activities of factors V, VII, protein C, antithrombin, and a disintegrin and metalloproteinase with thrombospondin-1 motifs (13th member of the family) (ADAMTS-13) decreased significantly in septic piglets compared with control. Histopathologic examination showed minor focal organ injuries including microvascular thrombi and necrosis in the kidney and liver of septic piglets. INTERPRETATIONS AND CONCLUSIONS: We established a 70-hour swine model of MRSA sepsis-induced coagulopathy with signs of consumptive coagulopathy, disseminated microvascular thrombosis, and early organ injuries with histological minor focal organ injuries. This model is clinically relevant to pediatric sepsis and can be used to study dysregulated host immune response and coagulopathy to infection, identify potential early biomarkers, and to test new therapeutics.

Secretion of von Willebrand Factor and Suppression of ADAMTS-13 Activity by Markedly High Concentration of Ferritin.

Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.

Is COVID-19-Induced Platelet Activation a Cause of Concern for Patients with Cancer?

Patients with cancer are more susceptible to be infected by SARS-CoV-2 and develop severe outcomes including ICU admittance, mechanical ventilator support, and a high rate of mortality. Like mid-to late-stage cancer, SARS-CoV-2 infection is associated with platelet hyperactivity, systemic inflammation, thrombotic complications, and coagulopathy. Platelets also promote cancer cell growth, survival in circulation, and angiogenesis at sites of metastases. In this article, we will discuss the potential for platelets in the development of systemic inflammation and thrombosis in SARS-CoV-2-infected patients with cancer, with the concern that the platelet-induced pathogenic events are likely magnified in cancer patients with COVID-19.

Modulating the rate of fibrin formation and clot structure attenuates microvascular thrombosis in systemic inflammation.

Systemic inflammation can lead to coagulopathy and disseminated intravascular coagulation (DIC). In prior studies, the recombinant A2 domain of human von Willebrand factor (VWF; A2 protein) attenuated DIC and decreased mortality in lipopolysaccharide (LPS)-treated mice. Here, we performed studies to dissect the mechanism by which the A2 protein moderates DIC. We used confocal microscopy to analyze the fibrin clot structure in plasma from healthy humans and endotoxemic mice, turbidity assays to examine fibrin polymerization, and a murine model for LPS-induced DIC and introduced a loss-of-function mutation into the A2 protein for fibrin. The mutation of the residue E1567 located in the α2 helix of the folded A2 domain of VWF inhibited binding activity for fibrin, possibly mapping a novel region containing a putative binding site for fibrin. The A2 protein increased the initial rate of change of fibrin polymerization, intercalated into the fibrin network, and modified the resultant clot structure in vitro. Furthermore, ex vivo experiments using plasma from mice with endotoxemia treated with the A2 protein revealed an increased rate of fibrin formation and an altered clot structure as compared with plasma from nontreated sick animals. Moreover, and in contrast to the A2 mutant, the A2 protein improved survival and reduced fibrin deposition and microvascular thrombosis in mice with endotoxemia-induced DIC. Importantly, in vivo and in vitro studies indicated that the A2 protein did not affect experimental thrombosis. Thus, we provide evidence for a novel treatment to attenuate systemic inflammation-induced coagulopathy/DIC via targeting fibrin formation, without an increased risk for bleeding.

Are Platelets the Primary Target of Aspirin's Remarkable Anticancer Activity?

Aspirin, when administered at low doses, has emerged as a powerful anticancer drug due to both chemopreventive activity against many forms of cancer and its ability to block metastases when administered postdiagnosis. Platelets, which are often elevated in circulation during the latter stages of cancer, are known to promote epithelial-mesenchymal transition, cancer cell growth, survival in circulation, and angiogenesis at sites of metastases. Low-dose aspirin has been demonstrated to block this procarcinogenic action of platelets. In this article, we present evidence that aspirin's unique ability to irreversibly inhibit platelet cyclooxygenase-1 is a key mechanism by which aspirin exerts anticancer activity.

In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion.

Essentials Platelet adhesion to von Willebrand factor (VWF) is critical for hemostasis and thrombosis. Whether VWF can undergo phosphorylation is unknown. Family with sequence similarity 20 kinase phosphorylates VWF A2 domain at S1517 and S1613. Phosphorylation of VWF and VWF A1A2A3 domain at S1613 enhances platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) mediates platelet adhesion and contributes to hemostasis at sites of vascular injury as well as to arterial thrombosis. The A1A2A3 domains of VWF contain important sites that differentially participate in supporting platelet adhesion. FAM20c (family with sequence similarity 20, member C) has emerged as a serine/threonine kinase, which phosphorylates extracellular proteins containing the S-X-E/pS motifs that are also found within the VWF A domains. This is of interest because we and others have shown that structural modifications within these A domains influence the ability of VWF to support platelet adhesion. Objective We assessed if VWF A domains can be phosphorylated and the functional consequence of phosphorylated VWF. Results Here, we show that FAM20c phosphorylated purified plasma VWF, VWF A1A2A3 protein, isolated A2 domain, but not A1 and A3 domain proteins, in vitro. FAM20c phosphorylated the isolated A2 domain at S1517 and S1613 within the S-X-E recognition motif, with S1613 being the major phosphorylation site. Mass spectrometry analysis of purified plasma VWF from healthy donors revealed several phosphorylation sites, including the S1613 in the A2 domain. VWF A1A2A3 domain protein phosphorylated at S1613 promoted stable platelet adhesion and microthrombi at high shear stress. Lastly, under high shear stress VWF treated with FAM20c and ATP robustly supported platelet adhesion, compared to VWF treated with FAM20c in the absence of ATP. Conclusion These outcomes indicate that VWF can be phosphorylated by FAM20c in vitro, and this novel post-translational modification enhances the adhesiveness of VWF to platelets.

The heterotrimeric G protein Gß1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gßγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gß1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gß1 revealed that Gß1 interacts with the PP1c α, ß, and γ1 isoforms. Purified PP1c bound to recombinant Gß1-GST protein, and PP1c co-immunoprecipitated with Gß1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gß1 complex, which correlated with an association of PP1c with phospholipase C ß3 (PLCß3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCß3. siRNA-mediated depletion of GNB1 (encoding Gß1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gßγ. Finally, disruption of PP1c-Gß1 complexes with myristoylated Gß1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gß1 protein enlists PP1c to modulate GPCR signaling in platelets.

Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway.

Treatment with dasatinib, a tyrosine kinase inhibitor, is associated with edema, pleural effusion, and pulmonary edema. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells-1 (HMEC-1) in vitro and in vivo. The permeability of HMEC-1 to fluorescein isothiocyante (FITC)-dextran increased in Transwell chambers within 5 min following the addition of therapeutic concentrations of dasatinib. The change in permeability was associated with increased activation of RhoA GTPase and its effector Rho-associated coiled-coil kinase 1(ROCK1). RhoA inhibitor C3 transferase almost completely inhibited dasatinib-induced increase in permeability. Under similar conditions, imatinib had no effect on permeability or activation of RhoA. Since integrin-induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impaired endothelial cell spreading in a concentration-dependent manner and induced disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibited tyrosine phosphorylation of p130 Crk-associated substrate (p130cas), paxillin and vinculin. These results suggest that the inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. Consistent with these findings, dasatinib-induced increase in the permeability was blocked by ROCK inhibitor y27632. In vivo administration of y27632, significantly inhibited the dasatinib-induced extravasation of Evans blue in mice and dasatinib-induced increase in microvascular permeability was attenuated in ROCK1-deficient mice. These findings suggest that ROCK inhibitors could serve as therapeutic modalities to ameliorate the dasatinib-induced pulmonary changes.

Disruption of protein complexes containing protein phosphatase 2B and Munc18c reduces the secretion of von Willebrand factor from endothelial cells.

Essentials Endothelial secretion of von Willebrand factor (VWF) promotes inflammation and thrombosis. We studied the role of protein phosphatase 2B (PP2B) and Munc18c protein complex in VWF secretion. Disruption of PP2B-Munc18c complex in endothelial cells reduced agonist-induced VWF secretion. PP2B-Munc18c complex represents a potential target for thrombotic and inflammatory conditions. SUMMARY: Background Aberrant secretion of von Willebrand factor (VWF) from endothelial cells contributes to inflammation and vascular thrombosis. Agonist-induced VWF secretion is facilitated by protein kinase and phosphatase-mediated signaling. Although the catalytic subunit of protein phosphatase 2B (PP2B-Aα) is targeted to the secretory machinery via an interaction with the vesicle trafficking protein Munc18c in endothelial cells, the functional relevance of this phosphatase complex is unclear. Objective To assess the contribution of the PP2B-Aα-Munc18c complex to endothelial VWF secretion. Results Here, we show that amino acids 120-130 of PP2B-Aα are important to support an interaction with Munc18c. A synthetic myristylated cell-permeable peptide, which is derived from amino acids 121-130 of PP2B-Aα, disrupted endogenous PP2B-Aα-Munc18c complexes in human umbilical vein endothelial cells, and decreased low-dose histamine-stimulated and thrombin-stimulated VWF secretion. Conclusion These studies indicate that PP2B-Aα-Munc18c complex supports agonist-induced VWF secretion, and suggest the potential of targeting this phosphatase complex in thrombotic and inflammatory conditions.

PAI1: a novel PP1-interacting protein that mediates human plasma's anti-apoptotic effect in endothelial cells.

Activation of apoptotic signalling in endothelial cells contributes to the detrimental effects of a variety of pathological stimuli. In investigating the molecular events underlying the anti-apoptotic effect of human plasma in cultured human endothelial cells, we unexpectedly uncovered a novel mechanism of apoptosis suppression by human plasma through an interaction between two previously unrelated proteins. Human plasma inhibited hypoxia-serum deprivation-induced apoptosis and stimulated BADS136 and AktS473 phosphorylation. Akt1 silencing reversed part (~52%) of the anti-apoptotic effect of human plasma, suggesting the existence of additional mechanisms mediating the anti-apoptotic effect other than Akt signalling. Human plasma disrupted the interaction of BAD with protein phosphatase 1 (PP1). Mass spectrometry identified fourteen PP1-interacting proteins induced by human plasma. Notably, a group of serine protease inhibitors including plasminogen activator inhibitor 1 (PAI1), a major inhibitor of fibrinolysis, were involved. Silencing of PAI1 attenuated the anti-apoptotic effect of human plasma. Furthermore, combined Akt1 and PAI1 silencing attenuated the majority of the anti-apoptotic effect of human plasma. We conclude that human plasma protects against endothelial cell apoptosis through sustained BAD phosphorylation, which is achieved by, at least in part, a novel interaction between PP1 with PAI1.

Unlocking Aspirin's Chemopreventive Activity: Role of Irreversibly Inhibiting Platelet Cyclooxygenase-1.

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirin's ability to irreversibly inhibit COX-1-mediated platelet activation, thereby blocking platelet-cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial-mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, ß-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirin's chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142-52. ©2016 AACR.

A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbß3 Integrin Signaling.

The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbß3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbß3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbß3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3ß. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity.

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer.

Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.

Platelets and Their Interactions with Other Immune Cells.

Platelets are anucleate blood cells, long known to be critically involved in hemostasis and thrombosis. In addition to their role in blood clots, increasing evidence reveals significant roles for platelets in inflammation and immunity. However, the notion that platelets represent immune cells is not broadly recognized in the field of Physiology. This article reviews the role of platelets in inflammation and immune responses, and highlights their interactions with other immune cells, including examples of major functional consequences of these interactions.

Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress.

The interaction between platelet receptor glycoprotein Ibα and the A1 domain of von Willebrand factor (VWF) mediates tethering/translocation of platelets to sites of vascular injury. Unexpectedly, we observed platelets translocating over A1A2A3 domains protein slower than on A1 domain at high shear stress. This observation suggests an additional interaction between A domains and an adhesive receptor. We investigated vimentin because we have data showing the interaction of vimentin with the A2 domain of VWF. Moreover, vimentin is expressed on the platelet surface. This novel interaction was analyzed by using purified VWF, recombinant proteins, anti-vimentin antibodies, parallel flow chamber adhesion assays, flow cytometry, and vimentin-deficient murine platelets. The active form of VWF bound to vimentin, and the purified A2 domain blocked that binding. The interaction of a gain-of-function A1A2A3 mutant with platelet was reduced using anti-vimentin antibody. Platelet adhesion to wild-type (WT) A1A2A3 protein, collagen, and fibrin(ogen) was inhibited (32-75%) by anti-vimentin antibody under high shear stress. Compared with WT mice, platelets from vimentin-deficient mice had a reduced flow-dependent adhesion to both collagen and purified murine VWF. Last, the vimentin knockout mice had a prolonged tail bleeding time. The results describe that platelet vimentin engages VWF during platelet adhesion under high shear stress.