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.

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.

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.

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.

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.

Signal transducer and activator of transcription 3 (STAT3) regulates collagen-induced platelet aggregation independently of its transcription factor activity.

BACKGROUND: Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. METHODS AND RESULTS: The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide-induced but not thrombin receptor-activating peptide- or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. CONCLUSIONS: These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.

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.

Cross-talk between serine/threonine protein phosphatase 2A and protein tyrosine phosphatase 1B regulates Src activation and adhesion of integrin αIIbß3 to fibrinogen.

Integrin α(IIb)ß(3) signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing α(IIb)ß(3) to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 α(IIb)ß(3) cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 α(IIb)ß(3) cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 α(IIb)ß(3) cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 α(IIb)ß(3) cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate α(IIb)ß(3) adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune α(IIb)ß(3) outside-in signaling.

Destabilization of the A1 domain in von Willebrand factor dissociates the A1A2A3 tri-domain and provokes spontaneous binding to glycoprotein Ibalpha and platelet activation under shear stress.

This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ibalpha (GPIbalpha). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIbalpha without ristocetin with an apparent K(D) of 85 +/- 34 nm, comparable with that of WT (36 +/- 12 nm) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s(-1) shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s(-1), platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIbalpha and alphaIIbbeta3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.

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.

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.

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.

Glutathione regulates integrin alpha(IIb)beta(3)-mediated cell adhesion under flow conditions.

The platelet integrin alpha(IIb)beta(3) mediates the final step of platelet aggregation that requires pre-activation through an inside-out signal initiated by agonists. Experiments conducted under static conditions using platelet-rich plasma show that platelet activation and adhesion activity of alpha(IIb)beta(3) are regulated by glutathione (GSH-GSSG) redox potential. However, it remains unclear as to whether GSH-GSSG exerts its regulatory role in platelets by direct targeting of alpha(IIb)beta(3) or intracellular signals that activate the integrin. A role of fluid shear stress is also not known. We examined the effects of GSH-GSSG on the adhesion of CHO cells expressing two HPA variants of human alpha(IIb)beta(3) to the immobilized fibrinogen and von Willebrand factor (VWF) under flow conditions. GSH-GSSG dose-dependently reduced the number of adherent cells to fibrinogen and VWF under 2.5 dyn/cm(2) of shear stress, a physical force calculated to be 110 dyne on platelets. GSH treatment also abolished the hyper-adhesion activity of cells expressing the Pro33 variant of alpha(IIb)beta(3). The inhibition was also observed with washed platelets. The data differ from the early observation that GSH enhanced platelet aggregation induced by sub-threshold concentrations of platelet agonists. The results suggest that GSH may have distinct effects on agonist-induced alpha(IIb)beta(3) activation and on the alpha(IIb)beta(3)-fibrinogen or alpha(IIb)beta(3)-VWF bonds when exposed to fluid shear stress. They further suggest that the HPA phenotype may be redox-regulated.