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Crosstalk between coagulation and innate immunity contributes to the progression of many diseases, including infection and cardiovascular disease. Venous thromboembolism (VTE), including pulmonary embolism and deep vein thrombosis (DVT), is among the most common causes of cardiovascular death. Here, we show that inflammasome activation and subsequent pyroptosis play an important role in the development of venous thrombosis. Using a flow restriction–induced mouse venous thrombosis model in the inferior vena cava (IVC), we show that deficiency of caspase-1, but not caspase-11, protected against flow restriction–induced thrombosis. Interleukin-1β expression increased in the IVC following ligation, indicating that inflammasome is activated during injury. Deficiency of gasdermin D (GSDMD), an essential mediator of pyroptosis, protected against restriction-induced venous thrombosis. After induction of venous thrombosis, fibrin was deposited in the veins of wild-type mice, as detected using immunoblotting with a monoclonal antibody that specifically recognizes mouse fibrin, but not in the caspase-1–deficient or GSDMD-deficient mice. Depletion of macrophages by gadolinium chloride or deficiency of tissue factor also protected against venous thrombosis. Our data reveal that tissue factor released from pyroptotic monocytes and macrophages following inflammasome activation triggers thrombosis.

Autophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis.

Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is one of the most common causes of cardiovascular death worldwide. Monocytic cells (including monocytes/macrophages) and their derived tissue factor have been reported to play important roles in the development of DVT. However, the mechanism by which monocytes contribute to the development DVT is not well elucidated. In this study, we reported a critical role of inflammasome activation and pyroptosis in the development of DVT. Using a flow restriction-induced mouse DVT model in the inferior vena cava, we show that deficiency of caspase-1 protected against flow restriction-induced DVT. Inflammasome activation leads to interleukin (IL)-1b and IL-18 maturation/release and pyroptosis. Recent studies show that caspases-1 cleaves Gasdermin D (GSDMD) and triggers pyroptosis-a form of programmed cell death with similar morphology to necrosis. We tested the hypothesis that GSDMD-dependent pyroptosis drives inflammasome-induced coagulation and DVT using Gsdmd-/- mice. Indeed, flow restriction-induced DVT was inhibited by GSDMD deficiency. After induction of DVT, fibrin was deposited in the vein as detected by Western blot with a monoclonal antibody that specifically recognizes mouse fibrin, which were inhibited in the caspase-1 deficient mice and GSDMD deficient mice. IL-1b was also increased in tissue of vein following induction of DVT, which was inhibited by caspase-1 or GSDMD deficiency. Following inflammasome activation, pyroptotic macrophages release tissue factor (TF), an essential initiator of coagulation cascades. Consistently, flow restriction-induced DVT was flow restriction-induced DVT inhibited in inducible TF deficient mice. Our data reveal a critical role of inflammasome activation and pyroptosis in the development of DVT. Disclosures No relevant conflicts of interest to declare.

Integrin activation (inside-out signaling) in platelets can be initiated by agonists such as von Willebrand factor (VWF) and thrombin. Here we show that a mitogen-activated protein kinase (MAPK), p38, plays an important role in the activation of integrin αIIbβ3 induced by VWF and thrombin. A dominant-negative mutant of p38, p38AF, inhibits αIIbβ3 activation induced by VWF binding to its receptor, the platelet glycoprotein Ib-IX (GPIb-IX), and p38 inhibitors diminish platelet aggregation induced by VWF or low-dose thrombin. The inhibitory effect of p38 inhibitor is unlikely to be caused by the previous suggested effect on cyclo-oxygenase, as inhibition also was observed in the presence of high concentrations of cyclo-oxygenase inhibitor, aspirin. VWF or thrombin induces p38 activation, which is inhibited in cGMP-dependent protein kinase (PKG)-knockout mouse platelets and PKG inhibitor-treated human platelets, indicating that activation of p38 is downstream from PKG in the signaling pathway. p38AF or p38 inhibitors diminish PKG-induced phosphorylation of extracellular stimuli-responsive kinase (ERK), which also is important in integrin activation. Thus, p38 plays an important role in mediating PKG-dependent activation of ERK. These data delineate a novel signaling pathway in which platelet agonists sequentially activate PKG, p38, and ERK pathways leading to integrin activation.

Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway.

Integrins play fundamental roles in many biological processes such as development, immunity, cancer, wound healing, hemostasis, and thrombosis. Integrin activation is essential for cell adhesion, spreading, survival, proliferation, and migration. Integrins are heterodimeric transmembrane glycoproteins composed of a and b subunits. The function of integrins is modulated by bi-directional transmembrane signaling: inside-out and outside-in signaling, which are mediated through the interactions between integrin a or b subunit cytoplasmic tails and intracellular proteins and can be regulated by many different biochemical signaling pathways. αIIbβ3 is a major integrin expressed in megakaryocytes and platelets. Antagonists of αIIbβ3 are potent anti-thrombotic drugs and new inhibitors targeting αIIbβ3 are under preclinical testing or large patient trials to treat acute coronary syndromes. Integrin αIIbβ3 activation involves binding of proteins, including talin, kindlins, Src kinase, and Gα13 to the cytoplasmic domain of its β subunit. To gain insight into αIIbβ3signaling and to identify new proteins that regulate αIIbβ3 activation, agarose beads conjugated with glutathione S-transferase (GST)-β3 integrin cytoplasmic domain fusion protein (GST-β3CD) or GST-αIIb integrin cytoplasmic domain fusion protein (GST-αIIbCD) were incubated with human platelet lysates. Proteins pulled down by the GST-β3CD or GST-αIIbCD beads were subjected to mass spectrometric analysis. We found that the GST-β3CD but not the GST-αIIbCD beads specifically pulled down a previously unreported protein, the vacuolar protein sorting-associated protein 33B (VPS33B), encoded by the VPS33B gene. To verify that VPS33B is in a complex with αIIbβ3, we infected Chinese hamster ovary (CHO) cells stably expressing αIIbβ3 (A5 cells) with an adenovirus containing FLAG epitope-tagged human VPS33B cDNA. Cells were allowed to adhere and spread on fibrinogen. FLAG-VPS33B exhibited a significant intracellular colocalization with aIIbb3. Immunoblotting analysis revealed a specific association of b3 with precipitated FLAG-VPS33B. The specific interaction was also confirmed by reciprocal immunoprecipitation using a specific β3antibody. Furthermore, in an in vitro binding assay, we were able to pull down VPS33B from the lysates of CHO cells overexpressing VPS33B by the GST-β3CD beads. The interaction appears to be direct, since we were also able to pull down purified FLAG-VPS33B with GST-β3CD and GST-β1CD proteins. The pulldown assay showed that the β3 fragment spanning residues 716~730 bound efficiently to FLAG-VPS33B. In contrast, the fragments containing either residues 729~762 or residues 744~762 fragments failed to do the same. More importantly, using the rabbit polyclonal antibody against VPS33B, we were able to coimmunoprecipitate endogenous b3 subunit with VPS33B from human platelet lysate. Thus, we identified a novel binding protein of integrin β3. Next, we investigated the role of VPS33B in integrin activation using a recombinant integrin activation model of CHO cells. Overexpression of VPS33B in CHO cells expressing αIIbβ3 (A5 cells) markedly potentiated cell spreading on fibrinogen and F-actin formation. To establish a role of VPS33B in integrin activation in platelets, we created a mouse model with megakaryocyte- and platelet-specific deletion of VPS33B. Platelets lacking VPS33B were defective in spreading on fibrinogen. VPS33B-/- platelets failed to support clot retraction. On the other hand, thrombin-induced fibrinogen binding to platelets and platelet aggregation were not affected by the loss of VPS33B. Collectively, these results demonstrate an essential role of VPS33B in αIIbβ3 outside-in signaling but is not requried for integrin inside-out signaling. We further demonstrate that VPS33B promotes αIIbβ3 outside-in signaling through RhoA and Rac1 activation, leading to clot retraction and cell spreading, respectively. Therefore, our results for the first time establish vesicle trafficking proteins as an important novel class of modifiers of integrin function in platelets and cells. Disclosures No relevant conflicts of interest to declare.

Abstract 93 Sepsis is a tremendous burden for health-care systems. Patients with sepsis often have low platelet counts, and septic patients with severe thrombocytopenia have a poor prognosis and higher mortality. However, the role of platelets in the pathogenesis of sepsis has not been well elucidated. We investigated the role of platelets in septic shock using a mouse model of lipopolysaccharide (LPS)-induced endotoxemia. Depletion of platelets by intraperitoneal injection of a rat anti-mouse GPIb monoclonal antibody increased mortality and aggravated organ failure in endotoxemic mice as evident by increases in plasma aminotransferase (ALT), aspartate aminotransferase (AST), Lactate dehydrogenase (LDH), and Creatine kinase (CK) concentrations, while transfusion of platelets reduced mortality. Increases in mortality rate in thrombocytopenic mice by LPS challenge was not due to inflammatory hemorrhage, because there was no significantly hemorrhage observed in brains and lungs from mice pre-treated with either control IgG or the anti-GPIba antibody and blood RBC and Hb concentrations between IgG pre-treated mice and the anti-GPIba antibody pre-treated mice were similar. TNF-a, which is produced mainly by macrophages in vivo, plays critical roles in the development of disseminated intravascular coagulation, acute respiratory distress syndrome and shock in sepsis. Our data indicate that plasma concentrations of proinflammatory cytokines, TNF-a and IL-6, were markedly increased by platelet depletion and decreased by platelet transfusion in the mice challenged with LPS. Effects of platelet depletion on TNF-a production were eliminated in the mice that macrophages were pre-depleted. Furthermore, LPS- or thrombin-activated platelets or releasates from activated platelets inhibited TNF-a and IL-6 production in macrophages in vitro. Inhibition of TNF-a and IL-6 production in macrophages by activated platelets was prevented by pre-incubation of platelets with a COX1 inhibitor aspirin. Moreover, platelets from wild type mice but not COX1 deficient mice inhibited LPS-induced TNF-a and IL-6 production in macrophages. Transfusion of COX1 deficient platelets failed to protect against endotoxemia. Washed platelets from wild-type mice or platelet releasates from thrombin-activated wild-type mice inhibited LPS-induced TNF-a and IL-6 production in macrophages lacking TXA2 receptor, TP, suggesting that a metabolite other than TXA2 is responsible for platelet inhibition of macrophage function. We found that stimulation of platelets with thrombin or LPS induced PGE2 production and pre-incubation of macrophages with an antagonist of PGE2 receptor EP4 reversed platelet inhibition on TNF-a and IL-6 production in macrophages. Our results indicate that platelets protect against septic shock by inhibiting macrophage-dependent inflammatory response via the COX1/PGE2/EP4 dependent pathway. Thus, these findings demonstrate a previously unappreciated role for platelets in septic shock and suggest that platelet transfusion may be effective in treating septic patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 5263 The Src family kinases (SFKs) play an essential role in collagen- and von Willebrand factor (vWF)-mediated platelet activation. However, the role of SFKs in G protein-coupled receptor (GPCR)-mediated platelet activation is not fully understood, and little is known about the molecular mechanisms by which SFKs are activated by GPCR stimulation. Here we demonstrate that SFKs are activated by the Gq and Gi pathways, respectively and SFKs play important roles in Gq- and Gi-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and Gq−/− platelets, and ADP-induced SFK phosphorylation was inhibited by the P2Y12 antagonist AR-C69931MX or P2Y12 knockout but was not affected by the P2Y1 antagonist MRS-2179. Lyn and Fyn were co-immunoprecipitated with Gi2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the Gi pathway activated via its receptor P2Y12. Furthermore, epinephrine also dose-dependently induced SFK phosphorylation in mouse platelets. A selective inhibitor of Src family kinase PP2 inhibited ADP-induced Akt phosphorylation, fibrinogen binding, and platelet aggregation. Thus, activation of Gi is sufficient to induce SFK activation, which plays an important role in Gi-dependent platelet activation. We further show that the thrombin receptor PAR4 peptide AYPGKF elicited SFK phosphorylation in P2Y12−/−, but not in Gq−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that Gq-dependent SFK phosphorylation is downstream from the Ca2+ signaling. The calcium ionophore, A23187-induced TXA2 synthesis, platelet aggregation and secretion were inhibited by pre-incubation of platelets with PP2. PAR4-induced TXA2 synthesis was also abolished by PP2. Moreover, PAR4-mediated granule secretion, integrin aIIbb3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by PP2 or a PKC inhibitor Ro-31-8220, but were completely abolished by Ro-31-8220 plus PP2 or dimethyl-BAPTA, suggesting that Ca2+/SFKs and PKC represent two parallel signaling pathways mediating Gq-dependent platelet activation. In summary, SFKs can be activated by Gq/Ca2+-dependent mechanisms and by Gi-dependent mechanisms, and SFKS play important roles in Gq- and Gi-dependent platelet activation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2200 Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

Abstract 3191 The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on Gi signaling activated primarily by secreted ADP through its receptor P2Y12. By using P2Y12 knockout mice combined with recombinant CHO models, we demonstrate a Gi-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y12 deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr308 and Ser473 in P2Y12 deficient platelets, demonstrating that there are P2Y12-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y12 deficient platelets, suggesting that Gi activation by thrombin or AYPGKF is dependent on ADP receptor P2Y12. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y12 deficient platelets is Gi independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y12 pathway, because CHO cells apparently do not express functional P2Y12. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y12 deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y12 dependent. In addition, P2Y12-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y12-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors. Disclosures: No relevant conflicts of interest to declare.

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.

Bacterial lipopolysaccharide (LPS) induces rapid thrombocytopenia, hypotension and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. Here we show that LPS stimulated platelet secretion of dense and alpha granules as indicated by ATP release and P-selectin expression, and thus enhanced platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including Toll-like receptor (TLR4), CD14, MD2, and MyD88. The effect of LPS on platelet activation was abolished by an anti-TLR4 blocking antibody or TLR4 knockout. Furthermore, LPS-induced potentiation of platelet aggregation and FeCl3-induced thrombus formation were abolished in MyD88 knockout mice. Importantly, TLR4 mediates LPS-induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was also abolished by inhibitors of nitric oxide synthase (NOS) and the cGMP-dependent protein kinase (PKG). Thus, LPS promotes platelet secretion and aggregation through a TLR4/MyD88 and cGMP/PKG-dependent pathway.

Although it was previously believed that the intracellular secondary messenger cGMP inhibits platelets, we have recently shown that cGMP-dependent protein kinase I (PKG I) in fact plays a stimulatory role in platelet activation. However, there are apparent differences between the PKG inhibitors and PKG I knockout in their effects on platelet activation. PKG inhibitors are more potent in inhibiting platelet activation than PKG I knockout. More importantly, although platelet secretion and aggregation induced by collagen were inhibited by PKG inhibitors, they are not significantly affected in PKG I knockout platelets. There are two types of PKG, PKG I and PKG II. PKG II has not been previously described in platelets. Here we show that PKG II mRNA is expressed in platelets using RT-PCR with primers specific for a C-terminal fragment of human PKG II cDNA. We further cloned the complete cDNA of human PKG II by RT-PCR using the purified human platelet mRNA as a template. Furthermore, PKG II from platelet lysates was pulled down by cGMP conjugated agarose beads and detected by western blot using a polyclonal antibody against PKG II. These data indicate that PKG II is expressed in platelets. To investigate the role of PKG II in platelet activation, washed wild type or PKG II knockout (PKG II−/−) mouse platelets in tyrode’s solution were exposed to platelet agonists. Platelet aggregation and ATP secretion induced by low concentrations of collagen were significantly reduced in PKG II deficient mice, indicating that PKG II plays important roles in collagen-induced platelet activation. PKG II−/− platelets also showed reduced aggregation and secretion to low dose of a thromboxane A2 (TXA2) analog, U46619. However, low dose thrombin-induced platelet activation was not negatively affected in PKG II−/− platelets, but was inhibited in PKG I−/− platelets. To evaluate the in vivo role of PKG II, we compared in vivo thrombus formation of wild type and PKG II knockout mice using the FeCl3-injured carotid artery thrombosis model. The time to the formation of stable thrombus in PKG II−/− mice (median, 420.0 seconds, n=15) is significantly prolonged compared to wild type mice (median, 321.0 seconds, n=15) (p=0.031). Tail-bleed time analysis also indicated a remarkably prolonged bleeding time in PKG II−/− mice (the median bleeding time was 73.50 seconds (n=18) in wild type mice, 454.50 seconds (n=20) in PKG II knockout mice) (p=0.0008). Thus, PKG II plays an important role in promoting platelet activation, thrombosis and hemostasis. PKG I and PKG II have differential roles in platelet activation induced by different platelet agonists.