Essentials Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor‐derived, tissue factor‐positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor‐derived, tissue factor‐positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs. Summary Background Cancer patients have an approximately 4‐fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor–derived, tissue factor–positive (TF⁺) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF⁺ tumor–derived MVs (TMVs) activate platelets in vitro and in mice. Materials and methods We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc‐3) and low (L3.6pl) levels of TF as models to study the effect of TF⁺ TMVs on platelets and thrombosis. Results and conclusions We found that both types of TF⁺ TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin‐dependent manner. Further, injection of BxPc‐3 TF⁺ TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF‐dependent manner. Importantly, BxPc‐3 TF⁺ TMV‐enhanced thrombosis was reduced in Par4‐deficient mice and in wild‐type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF⁺ TMV‐induced platelet activation contributes to thrombosis in cancer patients.

Objectives Behçet’s disease (BD) is a chronic systemic vasculitis. Thrombosis is a frequent and life-threatening complication. The pathogenesis of BD is poorly understood and evidence supporting a role for primed neutrophils in BD-associated thrombotic risk is scant. To respond to inflammatory insults, neutrophils release web-like structures, known as neutrophil extracellular traps (NETs), which are prothrombotic. We evaluated the role of NETs and markers of NETs in BD. Methods Blood samples were collected from patients with BD, according to the International Study Group Criteria for Behçet's disease, and healthy donors (HD). NET components, including cell-free DNA (CfDNA) and neutrophil enzymes myeloperoxidase (MPO), were assessed in serum or in purified neutrophils from patients with BD and HD. Results Patients with active BD had elevated serum cfDNA levels and MPO-DNA complexes compared with patients with inactive BD and to HD. In addition, levels of cfDNA and MPO-DNA complexes were significantly higher in patients with BD with vascular involvement compared with those without vascular symptoms. Purified neutrophils from patients with BD exhibited spontaneous NETosis compared with HD. Thrombin generation in BD plasma was significantly increased and positively correlated with the levels of MPO-DNA complexes and cfDNA. Importantly, DNAse treatment significantly decreased thrombin generation in BD plasma but not in HD plasma. In addition, biopsy materials obtained from patients with BD showed NETs production in areas of vasculitic inflammation and thrombosis. Conclusions Our data show that NETs and markers of NETS levels are elevated in patients with BD and contribute to the procoagulant state. Targeting NETs may represent a potential therapeutic target for the reduction or prevention of BD-associated thrombotic risk.

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Serine protease inhibitors, termed serpins, are key regulators in many biologic events. Protease nexin-1 (PN-1) is a serpin that is barely detectable in plasma but found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. It has a large inhibition spectrum because it is the most efficient tissue inhibitor of thrombin but also a powerful inhibitor of plasminogen activators and plasmin. It has a high affinity for glycosaminoglycans, such as heparan sulfates, which potentiate its activity toward thrombin and target it to the pericellular space. PN-1 has been previously largely described as a crucial regulator of the proteolytic activity in nerves and of central and peripheral nervous system function. In contrast, little was known about its involvement in hemostasis and vascular biology. This article reviews recent data underlining its emerging role as a key factor in the responses of vessels to injury. Indeed, studies of PN-1–deficient mice have established important antithrombotic and antifibrinolytic properties of this serpin that have heretofore gone unrecognized. The roles of PN-1 in the areas of hemostasis and thrombosis summarized here provide insights that may allow the development of drugs and treatment strategies to prevent or limit thrombotic disorders.

Objective—: The immune receptor homologue glycoprotein VI (GPVI)/FcR receptor γ chain complex is primarily responsible for platelet activation by collagen. There is growing evidence that optimal binding of GPVI to collagen depends on the assembly of GPVI dimers. The valence of GPVI on resting platelets needs to be clearly established because platelet avidity for collagen would be greater if GPVI is constitutively expressed as a dimer than as a monomer. Methods and Results—: Using a monoclonal antibody (9E18) that preferentially binds to GPVI dimers, we found that GPVI was maintained in a monomeric form on human resting platelets under the control of intraplatelet cAMP concentration. Activation by soluble agonists or von Willebrand factor induced a shift toward GPVI dimerization related to increased platelet adhesion to collagen. A correlation between platelet binding of 9E18 and P-selectin exposure was observed in patients experiencing coronary artery disease, and antagonists of the ADP receptor P2Y ₁₂ limited ADP-induced GPVI dimerization. Conclusion—: The rapid assembly of highly competent dimers of GPVI at sites of vascular lesion represents an important step in the sequence of events leading to platelet activation by collagen. GPVI dimers could represent a new marker to analyze platelet reactivity.