Illustrated State-of-the-Art Capsules of the ISTH 2024 Congress.

Here, we present a series of illustrated capsules from the State of the Art (SOA) speakers at the 2024 International Society on Thrombosis and Haemostasis Congress in Bangkok, Thailand. This year's Congress marks the first time that the International Society on Thrombosis and Haemostasis has held its flagship scientific meeting in Southeast Asia and is the first to be organized by an international Planning Committee. The Bangkok program will feature innovative science and clinical updates from around the world, reflecting the diversity and multidisciplinary growth of our field. In these illustrated SOA capsules, you will find an exploration of novel models of thrombosis and bleeding and biomaterial discoveries that can trigger or block coagulation. Thromboinflammation is now understood to drive many disease states, and the SOA speakers cover cellular and coagulation responses to COVID-19 and other infections. The theme of crosstalk between coagulation and inflammation expands with capsules on protein S signaling, complement, and fibrinolytic inhibitors. Novel agents for hemophilia and thrombosis prevention are introduced. Challenging clinical conditions are also covered, such as inherited platelet disorders and antiphospholipid antibody syndrome. The scientific program in Bangkok will also showcase the work of clinicians and scientists from all parts of the world and chronicle real-world challenges. For example, 2 SOA capsules address the diagnosis and management of von Willebrand disease in low-income settings. Take some time to browse through these short illustrated reviews; we're sure that you'll be entertained, educated, and inspired to further explore the world of thrombosis and hemostasis.

Increased risk of venous thromboembolism in young and middle-aged individuals with hereditary angioedema: a family study.

Hereditary angioedema (HAE) due to C1 inhibitor protein (C1-INH) deficiency was recently shown to be associated with increased risk of venous thromboembolism (VTE). This is the first national family study of HAE with the aim to determine the familial risk of VTE. The Swedish Multi-Generation Register was linked to the Swedish National Patient Register during the period 1964-2018. Only HAE patients with a validated diagnosis were included in the study and were linked to their family members. Hazard ratios (HRs) and 95% confidence intervals (CIs) for VTE were calculated for HAE patients compared with relatives without HAE. Among 2,006 individuals (from 276 pedigrees of 365 patients with HAE), 103 individuals were affected by VTE. In total 35 (9.6%) of HAE patients compared to 68 (4.1%) of non-HAE relatives were affected by VTE (p<0.001). The adjusted HR for VTE among HAE patients was 2.51 (95% CI 1.67-3.77). HAE patients were younger at the first VTE than their non-HAE relatives (mean age 51 versus 63 years, p<0.001). Before the age of 70 years the HR for VTE among HAE patients was 3.62 (95%CI 2.26-5.80). The HR for VTE for HAE patients born after 1964 was 8.29 (95%CI 2.90-23.71). The HR for VTE for HAE patients born 1964 or earlier was 1.82 (95%CI 1.14-2.91). HAE is associated with VTE among young and middle-aged individuals in Swedish families with HAE. The effect size of the association is in the order of other thrombophilias. We suggest that HAE may be considered a new rare thrombophilia.

Plasma kallikrein supports FXII-independent thrombin generation in mouse whole blood.

ABSTRACT: Plasma kallikrein (PKa) is an important activator of factor XII (FXII) of the contact pathway of coagulation. Several studies have shown that PKa also possesses procoagulant activity independent of FXII, likely through its ability to directly activate FIX. We evaluated the procoagulant activity of PKa using a mouse whole blood (WB) thrombin-generation (TG) assay. TG was measured in WB from PKa-deficient mice using contact pathway or extrinsic pathway triggers. PKa-deficient WB had significantly reduced contact pathway-initiated TG compared with that of wild-type controls and was comparable with that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway-initiated TG compared with wild-type controls. Consistent with the presence of FXII-independent functions of PKa, targeted blockade of PKa with either small molecule or antibody-based inhibitors significantly reduced contact pathway-initiated TG in FXII-deficient WB. Inhibition of activated FXII (FXIIa) using an antibody-based inhibitor significantly reduced TG in PKa-deficient WB, consistent with a PKa-independent function of FXIIa. Experiments using mice expressing low levels of tissue factor demonstrated that persistent TG present in PKa- and FXIIa-inhibited WB was driven primarily by endogenous tissue factor. Our work demonstrates that PKa contributes significantly to contact pathway-initiated TG in the complex milieu of mouse WB, and a component of this contribution occurs in an FXII-independent manner.

High plasma levels of C1-inhibitor are associated with lower risk of future venous thromboembolism.

BACKGROUND: C1-inhibitor (C1INH) is a broad-acting serine protease inhibitor with anticoagulant activity. The impact of C1INH plasma levels within the normal physiological range on risk of venous thromboembolism (VTE) is unknown. We assessed the association of plasma C1INH levels and VTE risk and evaluated the impact of C1INH on thrombin and plasmin generation in ex vivo assays. METHODS: A nested case-control study with 405 patients with VTE and 829 age- and sex-matched controls was derived from the Tromsø Study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE were estimated across plasma C1INH quartiles. Genetic regulation of C1INH was explored using quantitative trait loci analysis of whole exome sequencing data. The effect of plasma C1INH levels on coagulation was evaluated ex vivo by calibrated automated thrombography. RESULTS: Individuals with C1INH levels in the highest quartile had a lower risk of VTE (OR 0.68, 95% CI: 0.49-0.96) compared with those with C1INH in the lowest quartile. In subgroup analysis, the corresponding ORs were 0.60 (95% CI: 0.39-0.89) for deep vein thrombosis and 0.85 (95% CI: 0.52-1.38) for pulmonary embolism, respectively. No significant genetic determinants of plasma C1INH levels were identified. Addition of exogenous C1INH to normal human plasma reduced thrombin generation triggered by an activator of the intrinsic coagulation pathway, but not when triggered by an activator of the extrinsic coagulation pathway. CONCLUSIONS: High plasma levels of C1INH were associated with lower risk of VTE, and C1INH inhibited thrombin generation initiated by the intrinsic coagulation pathway ex vivo.

C1 inhibitor deficiency enhances contact pathway-mediated activation of coagulation and venous thrombosis.

C1 inhibitor (C1INH) is a multifunctional serine protease inhibitor that functions as a major negative regulator of several biological pathways, including the contact pathway of blood coagulation. In humans, congenital C1INH deficiency results in a rare episodic bradykinin-mediated swelling disorder called hereditary angioedema (HAE). Patients with C1INH deficiency-associated HAE (C1INH-HAE) have increased circulating markers of activation of coagulation. Furthermore, we recently reported that patients with C1INH-HAE had a moderate but significant increased risk of venous thromboembolism. To further investigate the impact of C1INH deficiency on activation of coagulation and thrombosis, we conducted studies using patient samples and mouse models. Plasmas from patients with C1INH-HAE had significantly increased contact pathway-mediated thrombin generation. C1INH-deficient mice, which have been used as a model of C1INH-HAE, had significantly increased baseline circulating levels of prothrombin fragment 1+2 and thrombin-antithrombin complexes. In addition, whole blood from C1INH-deficient mice supported significantly increased contact pathway-mediated thrombin generation. Importantly, C1INH-deficient mice exhibited significantly enhanced venous, but not arterial, thrombus formation. Furthermore, purified human C1INH normalized contact pathway-mediated thrombin generation and venous thrombosis in C1INH-deficient mice. These findings highlight a key role for endogenous C1INH as a negative regulator of contact pathway-mediated coagulation in humans and mice. Further, this work identifies endogenous C1INH as an important negative regulator of venous thrombus formation in mice, complementing the phenotype associated with C1INH-HAE.

Decreased Platelet Reactivity and Function in a Mouse Model of Human Pancreatic Cancer.

Cancer patients have increased thrombosis and bleeding compared with the general population. Cancer is associated with activation of both platelets and coagulation. Mouse models have been used to study the dysregulation of platelets and coagulation in cancer. We established a mouse model of pancreatic cancer in which tissue factor-expressing human pancreatic tumors (BxPC-3) are grown in nude mice. Tumor-bearing mice have an activated coagulation system and increased venous thrombosis compared to control mice. We also showed that tumor-derived, tissue factor-positive extracellular vesicles activated platelets ex vivo and in vivo. In this study, we determined the effect of tumors on a platelet-dependent arterial thrombosis model. Unexpectedly, we observed significantly reduced carotid artery thrombosis in tumor-bearing mice compared to controls. In addition, we observed significantly increased tail bleeding in tumor-bearing mice compared to controls. These results suggested that the presence of the tumor affected platelets. Indeed, tumor-bearing mice exhibited a significant decrease in platelet count and an increase in mean platelet volume and percentage of reticulated platelets, findings that are consistent with increased platelet turnover. Levels of the platelet activation marker platelet factor 4 were also increased in tumor-bearing mice. We also observed decreased platelet receptor expression in tumor-bearing mice and reduced levels of active αIIb/ß3 integrin in response to PAR4 agonist peptide and convulxin in platelets from tumor-bearing mice compared with platelets from control mice. In summary, our study suggests that in tumor-bearing mice there is chronic platelet activation, leading to thrombocytopenia, decreased receptor expression, and impaired platelet adhesive function.

Thrombin-mediated activation of PAR1 enhances doxorubicin-induced cardiac injury in mice.

The chemotherapeutic drug doxorubicin is cardiotoxic and can cause irreversible heart failure. In addition to being cardiotoxic, doxorubicin also induces the activation of coagulation. We determined the effect of thrombin-mediated activation of protease-activated receptor 1 (PAR1) on doxorubicin-induced cardiac injury. Administration of doxorubicin to mice resulted in a significant increase in plasma prothrombin fragment 1+2, thrombin-antithrombin complexes, and extracellular vesicle tissue factor activity. Doxorubicin-treated mice expressing low levels of tissue factor, but not factor XII-deficient mice, had reduced plasma thrombin-antithrombin complexes compared to controls. To evaluate the role of thrombin-mediated activation of PAR1, transgenic mice insensitive to thrombin (Par1R41Q) or activated protein C (Par1R46Q) were subjected to acute and chronic models of doxorubicin-induced cardiac injury and compared with Par1 wild-type (Par1+/+) and PAR1 deficient (Par1-/-) mice. Par1R41Q and Par1-/- mice, but not Par1R46Q mice, demonstrated similar reductions in the cardiac injury marker cardiac troponin I, preserved cardiac function, and reduced cardiac fibrosis compared to Par1+/+ controls after administration of doxorubicin. Furthermore, inhibition of Gαq signaling downstream of PAR1 with the small molecule inhibitor Q94 significantly preserved cardiac function in Par1+/+ mice, but not in Par1R41Q mice subjected to the acute model of cardiac injury when compared to vehicle controls. In addition, mice with PAR1 deleted in either cardiomyocytes or cardiac fibroblasts demonstrated reduced cardiac injury compared to controls. Taken together, these data suggest that thrombin-mediated activation of PAR1 contributes to doxorubicin-induced cardiac injury.

A novel mouse whole blood thrombin generation assay sensitive to FXI- and FIX-mediated amplification of coagulation.

Thrombin generation (TG) assays serve as a valuable tool to study the amplifying roles of intrinsic pathway factors in human coagulation and provide functional insights into the increased bleeding observed in individuals deficient in factors (F) XI, IX, or VIII. Mice are used extensively in hemostasis research owing to the availability of coagulation factor-deficient mice. However, phenotypic differences between mouse and human TG have become apparent. In this study, we describe a novel, calibrated mouse whole blood (WB) TG assay used to assess the amplifying roles of intrinsic pathway factors in mouse coagulation. WB- and plasma-TG was triggered with either silica or tissue factor (TF) in samples from wild-type mice and mice deficient for FXII, FXI, or FIX. Expectedly, silica-triggered WB-TG and platelet-poor plasma (PPP)-TG were significantly reduced by deficiencies for FXII, FXI, or FIX. FXII deficiency had no effect on WB-TG or PPP-TG when triggered with TF. However, FXI deficiency resulted in significantly reduced WB-TG triggered by low concentrations of TF but had no effect on TF-triggered PPP-TG. FIX deficiency profoundly reduced WB-TG when triggered by low or high concentrations of TF whereas TG in PPP or platelet-rich plasma was only moderately reduced under these conditions. In conclusion, we have developed a novel mouse WB-TG assay with enhanced sensitivity to FXI- and FIX-dependent amplification of coagulation compared with an established plasma-TG assay. The enhanced sensitivity of WB-TG to FXI and FIX-dependent amplification of coagulation suggests an important role of blood cells in this process.

Anticoagulant SERPINs: Endogenous Regulators of Hemostasis and Thrombosis.

Appropriate activation of coagulation requires a balance between procoagulant and anticoagulant proteins in blood. Loss in this balance leads to hemorrhage and thrombosis. A number of endogenous anticoagulant proteins, such as antithrombin and heparin cofactor II, are members of the serine protease inhibitor (SERPIN) family. These SERPIN anticoagulants function by forming irreversible inhibitory complexes with target coagulation proteases. Mutations in SERPIN family members, such as antithrombin, can cause hereditary thrombophilias. In addition, low plasma levels of SERPINs have been associated with an increased risk of thrombosis. Here, we review the biological activities of the different anticoagulant SERPINs. We further consider the clinical consequences of SERPIN deficiencies and insights gained from preclinical disease models. Finally, we discuss the potential utility of engineered SERPINs as novel therapies for the treatment of thrombotic pathologies.

Effect of combining aspirin and rivaroxaban on atherosclerosis in mice.

BACKGROUND AND AIMS: The platelet inhibitor aspirin reduces inflammation and atherosclerosis in both apolipoprotein E deficient (apoE-/-) mice and low-density lipoprotein receptor deficient (Ldlr-/-) mice. Similarly, the factor Xa inhibitor rivaroxaban reduces atherosclerosis in both apoE-/- and Ldlr-/- mice. We tested the hypothesis that the combination of aspirin and rivaroxaban reduces atherosclerosis in mice to a greater extent than either agent alone. METHODS: Male Ldlr-/- mice were fed a western-type diet for 12 weeks to induce atherosclerosis. Cohorts of mice received aspirin in the water and/or rivaroxaban in the diet. Atherosclerosis and lesion composition were measured in the aortic sinus and the aorta. Expression of 55 proteins in the aorta and plasma was determined using multiplex ELISA assays. RESULTS: Aspirin alone, rivaroxaban alone, and the combination of both agents significantly reduced atherosclerosis in the Ldlr-/- mice compared with control Ldlr-/- mice fed a western-type diet. However, there were no significant differences in atherosclerosis in the group receiving aspirin and rivaroxaban compared with the groups that received aspirin or rivaroxaban alone. Aspirin, rivaroxaban and the combination reduced macrophage content and apoptosis in the lesions compared with controls but there was no difference between the three treatment groups. We observed statistically significant changes in the expression of a small number of proteins in the aorta and plasma in mice treated with aspirin and/or rivaroxaban. CONCLUSIONS: Contrary to our expectation, the combination of aspirin and rivaroxaban did not further reduce atherosclerosis in Ldlr-/- mice beyond the level observed with each agent alone.

Genetic deletion of platelet PAR4 results in reduced thrombosis and impaired hemostatic plug stability.

BACKGROUND: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. OBJECTIVE: We aimed to generate a mouse line with a megakaryocyte/platelet-specific deletion of PAR4 (PAR4fl/fl ;PF4Cre+ ) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. METHODS: Platelets from PAR4fl/fl ;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet-specific deletion of PAR4 or wild-type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. RESULTS: PAR4fl/fl ;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4-/- and PAR4fl/fl ;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4-/- mice and PAR4fl/fl ;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild-type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. CONCLUSION: We generated a novel PAR4fl/fl ;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

Tissue factor expression, extracellular vesicles, and thrombosis after infection with the respiratory viruses influenza A virus and coronavirus.

Tissue factor (TF) is induced in a variety of cell types during viral infection, which likely contributes to disseminated intravascular coagulation and thrombosis. TF-expressing cells also release TF-positive extracellular vesicles (EVs) into the circulation that can be measured using an EVTF activity assay. This review summarizes studies that analyze TF expression, TF-positive EVs, activation of coagulation, and thrombosis after infection with influenza A virus (IAV) and coronaviruses (CoVs), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). The current pandemic of coronavirus disease 2019 (COVID-19) is caused by infection with SARS-CoV-2. Infection of mice with IAV increased TF expression in lung epithelial cells as well as increased EVTF activity and activation of coagulation in the bronchoalveolar lavage fluid (BALF). Infection of mice with MERS-CoV, SARS-CoV, and SARS-CoV-2 also increased lung TF expression. Single-cell RNA sequencing analysis on the BALF from severe COVID-19 patients revealed increased TF mRNA expression in epithelial cells. TF expression was observed in peripheral blood mononuclear cells infected with SARS-CoV. TF was also expressed by peripheral blood mononuclear cells, monocytes in platelet-monocyte aggregates, and neutrophils isolated from COVID-19 patients. Elevated circulating EVTF activity was observed in severe IAV and COVID-19 patients. Importantly, EVTF activity was associated with mortality in severe IAV patients and with plasma D-dimer, severity, thrombosis, and mortality in COVID-19 patients. These studies strongly suggest that increased TF expression in patients infected with IAV and pathogenic CoVs contributes to thrombosis.

Comparison of the coagulopathies associated with COVID-19 and sepsis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with activation of coagulation that mainly presents as thrombosis. Sepsis is also associated with activation of coagulation that mainly presents as disseminated intravascular coagulation. Many studies have reported increased levels of plasma d-dimer in patients with COVID-19 that is associated with severity, thrombosis, and mortality. OBJECTIVES: The aim of this study was to compare levels of circulating extracellular vesicle tissue factor (EVTF) activity and active plasminogen activator inhibitor 1 (PAI-1) in plasma from patients with COVID-19 or sepsis. METHODS: We measured levels of d-dimer, EVTF activity, and active PAI-1 in plasma samples from patients with COVID-19 (intensive care unit [ICU], N = 15; and non-ICU, N = 20) and patients with sepsis (N = 35). RESULTS: Patients with COVID-19 had significantly higher levels of d-dimer, EVTF activity, and active PAI-1 compared with healthy controls. Patients with sepsis had significantly higher levels of d-dimer and EVTF activity compared with healthy controls. Levels of d-dimer were significantly lower in patients with COVID-19 compared with patients with sepsis. Levels of EVTF activity were significantly higher in ICU patients with COVID-19 compared with patients with sepsis. Levels of active PAI-1 were significantly higher in patients with COVID-19 compared with patients with sepsis. CONCLUSIONS: High levels of both EVTF activity and active PAI-1 may promote thrombosis in patients with COVID-19 due to simultaneous activation of coagulation and inhibition of fibrinolysis. The high levels of active PAI-1 in patients with COVID-19 may limit plasmin degradation of crosslinked fibrin and the release of d-dimer. This may explain the lower levels of D-dimer in patients with COVID-19 compared with patients with sepsis.