TFPIα anticoagulant function is highly dependent on protein S in vivo.

Tissue factor pathway inhibitor α (TFPIα) is the major physiological regulator of the initiation of blood coagulation. In vitro, TFPIα anticoagulant function is enhanced by its cofactor, protein S. To define the role of protein S enhancement in TFPIα anticoagulant function in vivo, we blocked endogenous TFPI in mice using a monoclonal antibody (14D1). This caused a profound increase in fibrin deposition using the laser injury thrombosis model. To explore the role of plasma TFPIα in regulating thrombus formation, increasing concentrations of human TFPIα were coinjected with 14D1, which dose-dependently reduced fibrin deposition. Inhibition of protein S cofactor function using recombinant C4b-binding protein ß chain significantly reduced the anticoagulant function of human TFPIα in controlling fibrin deposition. We report an in vivo model that is sensitive to the anticoagulant properties of the TFPIα-protein S pathway and show the importance of protein S as a cofactor in the anticoagulant function of TFPIα in vivo.

The TFPIα C-terminal tail is essential for TFPIα-FV-short-protein S complex formation and synergistic enhancement of TFPIα.

BACKGROUND: For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear. OBJECTIVES: To determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement. METHODS: TFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1-249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation. RESULTS: While WT TFPIα and TFPIα K3m bound FV-short with high affinity (Kd∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively. CONCLUSION: Full TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.

Illustrated State-of-the-Art Capsules of the ISTH 2023 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) took place in person in Montréal, Canada, from June 24-28, 2023. The conference, held annually, highlighted cutting-edge advances in basic, translational, population and clinical sciences relevant to the Society. As for all ISTH congresses, we offered a special, congress-specific scientific theme; this year, the special theme was immunothrombosis. Certainly, over the last few years, COVID-19 infection and its related thrombotic and other complications have renewed interest in the concepts of thromboinflammation and immunothrombosis; namely, the relationship between inflammation, infection and clotting. Other main scientific themes of the Congress included Arterial Thromboembolism, Coagulation and Natural Anticoagulants, Diagnostics and Omics, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostatic System in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. Among other sessions, the program included 28 State-of-the-Art (SOA) sessions with a total of 84 talks given by internationally recognized leaders in the field. SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the Congress.

The role of CD8+ T-cell clones in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

Platelet-Neutrophil Crosstalk in Thrombosis.

Platelets are essential for the formation of a haemostatic plug to prevent bleeding, while neutrophils are the guardians of our immune defences against invading pathogens. The interplay between platelets and innate immunity, and subsequent triggering of the activation of coagulation is part of the host system to prevent systemic spread of pathogen in the blood stream. Aberrant immunothrombosis and excessive inflammation can however, contribute to the thrombotic burden observed in many cardiovascular diseases. In this review, we highlight how platelets and neutrophils interact with each other and how their crosstalk is central to both arterial and venous thrombosis and in COVID-19. While targeting platelets and coagulation enables efficient antithrombotic treatments, they are often accompanied with a bleeding risk. We also discuss how novel approaches to reduce platelet-mediated recruitment of neutrophils could represent promising therapies to treat thrombosis without affecting haemostasis.

The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling.

The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing both VWF-GPIbGGand collagen-GPVI signaling events in platelets.

Fibrinogen-mimicking, multiarm nanovesicles for human thrombus-specific delivery of tissue plasminogen activator and targeted thrombolytic therapy.

Clinical use of tissue plasminogen activator (tPA) in thrombolytic therapy is limited by its short circulation time and hemorrhagic side effects. Inspired by fibrinogen binding to activated platelets, we report a fibrinogen-mimicking, multiarm nanovesicle for thrombus-specific tPA delivery and targeted thrombolysis. This biomimetic system is based on the lipid nanovesicle coated with polyethylene glycol (PEG) terminally conjugated with a cyclic RGD (cRGD) peptide. Our experiments with human blood demonstrated its highly selective binding to activated platelets and efficient tPA release at a thrombus site under both static and physiological flow conditions. Its clot dissolution time in a microfluidic system was comparable to that of free tPA. Furthermore, we report a purpose-built computational model capable of simulating targeted thrombolysis of the tPA-loaded nanovesicle and with a potential in predicting the dynamics of thrombolysis in physiologically realistic scenarios. This combined experimental and computational work presents a promising platform for development of thrombolytic nanomedicines.

Activated αIIbß3 on platelets mediates flow-dependent NETosis via SLC44A2.

Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Ibα-dependent platelet 'priming' induces integrin αIIbß3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet αIIbß3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated αIIbß3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil crosstalk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.

Platelets in our blood form clots over sites of injury to stop us from bleeding. Blood clots can also occur in places where they are not needed, such as deep veins in our legs or other regions of the body. Developing such clots ­ also known as deep vein thrombosis (or DVT for short) ­ is one of the most common cardiovascular diseases and a major cause of death. Although certain inherited factors have been linked to DVT, the underlying mechanisms of the disease remain poorly understood. In addition to platelets, the pathological (or dangerous) clots that cause DVT also contain immune cells called neutrophils which fight off bacterial infections. Platelets are recruited to the wall of the vein by a protein called "von Willebrand Factor" (or VWF for short). However, it remained unclear how these recruited platelets interact with neutrophils and whether this promotes the onset of DVT. To answer this question, Constantinescu-Bercu et al. used a device that mimics the flow of blood to study how human platelets change when they are exposed to VWF. This revealed that VWF 'primes' the platelets to interact with neutrophils via a protein called integrin α_IIbß₃. Further experiments showed that integrin α_IIbß₃ binds to a protein on the surface of neutrophils called SLC44A2. Once the neutrophils interacted with the 'primed' platelets, they started making traps which increased the size of the blood clot by capturing other blood cells and proteins. Finally, Constantinescu-Bercu et al. studied a genetic variant of the SLC44A2 protein which is found in 22% of people and is associated with a lower risk of developing DVT. This genetic mutation caused SLC44A2 to interact with 'primed' platelets more weakly, which may explain why people with this genetic variant are protected from getting DVT. These findings suggest that blocking the interaction between 'primed' platelets and neutrophils could reduce the risk of DVT. Although current treatments for DVT can prevent patients from forming dangerous blood clots, they can also cause severe bleeding. Since neutrophils are not crucial for normal blood clots to form at the site of injury, drugs targeting SLC44A2 could inhibit inappropriate clotting without causing excess bleeding.

The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex.

BACKGROUND: Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive. OBJECTIVES: To elucidate the nature of the phospholipid-dependent interactions among APC, protein S, and FVa. METHODS: We evaluated binding of active site blocked APC to phospholipid-coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally. RESULTS: Activated protein C alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding (>14-fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara (W1920R), an APC-resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex. CONCLUSIONS: FVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions among FVa, APC, and protein S are essential for its cofactor function.

Defective fibrin deposition and thrombus stability in Bambi-/- mice are mediated by elevated anticoagulant function.

BACKGROUND: Bone morphogenetic and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the type I transforming growth factor- ß (TGF-ß) receptor family that is present on both platelets and endothelial cells (ECs). Bambi-deficient mice exhibit reduced hemostatic function and thrombus stability characterized by an increased embolization. OBJECTIVE: We aimed to delineate how BAMBI influences endothelial function and thrombus stability. METHODS: Bambi-deficient mice were subjected to the laser-induced thrombosis model where platelet and fibrin accumulation was evaluated. Expression of thrombomodulin and tissue factor pathway inhibitor (TFPI) was also assessed in these mice. RESULTS: Thrombus instability in Bambi-/- mice was associated with a profound defect in fibrin deposition. Injection of hirudin into Bambi+/+ mice prior to thrombus formation recapitulated the Bambi-/- thrombus instability phenotype. In contrast, hirudin had no additional effect upon thrombus formation in Bambi-/- mice. Deletion of Bambi in ECs resulted in mice with defective thrombus stability caused by decreased fibrin accumulation. Increased levels of the anticoagulant proteins TFPI and thrombomodulin were detected in Bambi-/- mouse lung homogenates. Endothelial cells isolated from Bambi-/- mouse lungs exhibited enhanced ability to activate protein C due to elevated thrombomodulin levels. Blocking thrombomodulin and TFPI in vivo fully restored fibrin accumulation and thrombus stability in Bambi-/- mice. CONCLUSIONS: We demonstrate that endothelial BAMBI influences fibrin generation and thrombus stability by modulating thrombomodulin and TFPI anticoagulant function of the endothelium; we also highlight the importance of these anticoagulant proteins in the laser-induced thrombosis model.

Artificial MiRNA Knockdown of Platelet Glycoprotein lbα: A Tool for Platelet Gene Silencing.

In recent years, candidate genes and proteins implicated in platelet function have been identified by various genomic approaches. To elucidate their exact role, we aimed to develop a method to apply miRNA interference in platelet progenitor cells by using GPIbα as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIbα (siGPIBAs), we developed artificial miRNAs (miGPIBAs), which were tested in CHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introduction of siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown of GPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAs to miGPIBAs resulted in reduced silencing efficiency, which could however be circumvented by tandem integration of two hairpins targeting different regions of GPIBA mRNA where 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBA construct displayed a significant decrease in their ability to aggregate characterized by lower aggregate numbers and size compared to control CHO GPIb-IX cells. More importantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMI cells that exhibited morphological changes associated with actin organization. In conclusion, we here report the successful use of miRNA technology to silence a platelet protein in megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, we believe that artificial miRNAs are suitable tools to unravel the role of a protein of interest in stem cells, megakaryocytes and platelets, thereby expanding their application to novel fields of basic and translational research.

Vessel wall BAMBI contributes to hemostasis and thrombus stability.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the transforming growth factor-ß superfamily, and is highly expressed in platelets and endothelial cells. We previously demonstrated its positive role in thrombus formation using a zebrafish thrombosis model. In the present study, we used Bambi-deficient mice and radiation chimeras to evaluate the function of this receptor in the regulation of both hemostasis and thrombosis. We show that Bambi(-/-) and Bambi(+/-) mice exhibit mildly prolonged bleeding times compared with Bambi(+/+) littermates. In addition, using 2 in vivo thrombosis models in mesenterium or cremaster muscle arterioles, we demonstrate that Bambi-deficient mice form unstable thrombi compared with Bambi(+/+) mice. No defects in thrombin generation in Bambi(-/-) mouse plasma could be detected ex vivo. Moreover, the absence of BAMBI had no effect on platelet counts, platelet activation, aggregation, or platelet procoagulant function. Similar to Bambi(-/-) mice, Bambi(-/-) transplanted with Bambi(+/+) bone marrow formed unstable thrombi in the laser-induced thrombosis model that receded more rapidly than thrombi that formed in Bambi(+/+) mice receiving Bambi(-/-) bone marrow transplants. Taken together, these results provide strong evidence for an important role of endothelium rather than platelet BAMBI as a positive regulator of both thrombus formation and stability.