Viper venoms drive the macrophages and hepatocytes to sequester and clear platelets: novel mechanism and therapeutic strategy for venom-induced thrombocytopenia.

Venomous snakebites cause clinical manifestations that range from local to systemic and are considered a significant global health challenge. Persistent or refractory thrombocytopenia has been frequently reported in snakebite patients, especially in cases caused by viperidae snakes. Viper envenomation-induced thrombocytopenia may persist in the absence of significant consumption coagulopathy even after the antivenom treatment, yet the mechanism remains largely unknown. Our study aims to investigate the mechanism and discover novel therapeutic targets for coagulopathy-independent thrombocytopenia caused by viper envenomation. Here we found that patients bitten by Protobothrops mucrosquamatus and Trimeresurus stejnegeri, rather than Naja. atra may develop antivenom-resistant and coagulopathy-independent thrombocytopenia. Crude venoms and the derived C-type lectin-like proteins from these vipers significantly increased platelet surface expression of neuraminidase and platelet desialylation, therefore led to platelet ingestion by both macrophages and hepatocytes in vitro, and drastically decreased peripheral platelet counts in vivo. Our study is the first to demonstrate that desialylation-mediated platelet clearance is a novel mechanism of viper envenomation-induced refractory thrombocytopenia and C-type lectin-like proteins derived from the viper venoms contribute to snake venom-induced thrombocytopenia. The results of this study suggest the inhibition of platelet desialylation as a novel therapeutic strategy against viper venom-induced refractory thrombocytopenia.

The 14-3-3ζ-c-Src-integrin-ß3 complex is vital for platelet activation.

Several adaptor molecules bind to cytoplasmic tails of ß-integrins and facilitate bidirectional signaling, which is critical in thrombosis and hemostasis. Interfering with integrin-adaptor interactions spatially or temporally to inhibit thrombosis without affecting hemostasis is an attractive strategy for the development of safe antithrombotic drugs. We show for the first time that the 14-3-3ζ-c-Src-integrin-ß3 complex is formed during platelet activation. 14-3-3ζ-c-Src interaction is mediated by the -PIRLGLALNFSVFYYE- fragment (PE16) on the 14-3-3ζ and SH2-domain on c-Src, whereas the 14-3-3ζ-integrin-ß3 interaction is mediated by the -ESKVFYLKMKGDYYRYL- fragment (EL17) on the 14-3-3ζ and -KEATSTF- fragment (KF7) on the ß3-integrin cytoplasmic tail. The EL17-motif inhibitor, or KF7 peptide, interferes with the formation of the 14-3-3ζ-c-Src-integrin-ß3 complex and selectively inhibits ß3 outside-in signaling without affecting the integrin-fibrinogen interaction, which suppresses thrombosis without causing significant bleeding. This study characterized a previously unidentified 14-3-3ζ-c-Src-integrin-ß3 complex in platelets and provided a novel strategy for the development of safe and effective antithrombotic treatments.

The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

CD8+ T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder characterized by autoantibodies targeting platelet surface proteins, most commonly GPIIbIIIa (αIIbß3 integrin), leading to platelet destruction. Recently, CD8(+) cytotoxic T-lymphocytes (CTLs) targeting platelets and megakaryocytes have also been implicated in thrombocytopenia. Because steroids are the most commonly administered therapy for ITP worldwide, we established both active (immunized splenocyte engraftment) and passive (antibody injection) murine models of steroid treatment. Surprisingly, we found that, in both models, CD8(+) T cells limited the severity of the thrombocytopenia and were required for an efficacious response to steroid therapy. Conversely, CD8(+) T-cell depletion led to more severe thrombocytopenia, whereas CD8(+) T-cell transfusion ameliorated thrombocytopenia. CD8(+) T-regulatory cell (Treg) subsets were detected, and interestingly, dexamethasone (DEX) treatment selectively expanded CD8(+) Tregs while decreasing CTLs. In vitro coculture studies revealed CD8(+) Tregs suppressed CD4(+) and CD19(+) proliferation, platelet-associated immunoglobulin G generation, CTL cytotoxicity, platelet apoptosis, and clearance. Furthermore, we found increased production of anti-inflammatory interleukin-10 in coculture studies and in vivo after steroid treatment. Thus, we uncovered subsets of CD8(+) Tregs and demonstrated their potent immunosuppressive and protective roles in experimentally induced thrombocytopenia. The data further elucidate mechanisms of steroid treatment and suggest therapeutic potential for CD8(+) Tregs in immune thrombocytopenia.

Maternal anti-platelet ß3 integrins impair angiogenesis and cause intracranial hemorrhage.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against ß3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-ß3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-ß3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

Plant food delphinidin-3-glucoside significantly inhibits platelet activation and thrombosis: novel protective roles against cardiovascular diseases.

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbß3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbß3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.