Angiopoietin2 is associated with coagulation activation and tissue factor expression in extracellular vesicles in COVID-19.

Coagulation activation in immunothrombosis involves various pathways distinct from classical hemostasis, offering potential therapeutic targets to control inflammation-induced hypercoagulability while potentially sparing hemostasis. The Angiopoietin/Tie2 pathway, previously linked to embryonic angiogenesis and sepsis-related endothelial barrier regulation, was recently associated with coagulation activation in sepsis and COVID-19. This study explores the connection between key mediators of the Angiopoietin/Tie2 pathway and coagulation activation. The study included COVID-19 patients with hypoxia and healthy controls. Blood samples were processed to obtain platelet-free plasma, and frozen until analysis. Extracellular vesicles (EVs) in plasma were characterized and quantified using flow cytometry, and their tissue factor (TF) procoagulant activity was measured using a kinetic chromogenic method. Several markers of hemostasis were assessed. Levels of ANGPT1, ANGPT2, and soluble Tie2 correlated with markers of coagulation and platelet activation. EVs from platelets and endothelial cells were increased in COVID-19 patients, and a significant increase in TF+ EVs derived from endothelial cells was observed. In addition, ANGPT2 levels were associated with TF expression and activity in EVs. In conclusion, we provide further evidence for the involvement of the Angiopoietin/Tie2 pathway in the coagulopathy of COVID-19 mediated in part by release of EVs as a potential source of TF activity.

Fewer severe infections with tranexamic acid in patients with hematologic malignancies.

Background: Tranexamic acid (TXA) is an antifibrinolytic agent that reduces bleeding in a multitude of clinical settings from postpartum hemorrhage to trauma. TXA may have clinical effects unrelated to bleeding; plasminogen, the target of TXA, alters immune responses, and TXA appears to decrease the risk of infection in patients undergoing cardiac surgery, as well as joint arthroplasty. Objectives: To address whether TXA alters rates of infection and inflammatory outcomes in patients with hematologic malignancies. Methods: We performed a post hoc analysis of outcomes of patients randomized to receive either TXA or placebo in the double-blinded, multicenter American Trial to Evaluate Tranexamic Acid Therapy in Thrombocytopenia (Clinicaltrials.gov identifier: NCT02578901). Results: TXA did not change the overall rate of infections, but the rate of severe infections (Common Toxicology Criteria for Adverse Events grade 3+) was lower in patients who received TXA compared with the placebo group. Patients who experienced grade 3+ infections had higher rates of World Health Organization grade 2+ bleeding and red blood cell transfusion requirements than patients who did not experience a grade 3+ infection, irrespective of treatment group. TXA did not impact other inflammatory outcomes such as mucositis, rash, or graft vs host disease. Conclusion: Patients with hematologic malignancies who received TXA had less severe infections than those who received placebo with no difference in overall rate of infection or other inflammatory outcomes. Further investigation is needed on the impact of TXA on infections in this population.

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

Plasma kallikrein (PKa) is an important activator of factor (F)XII 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 to wild-type controls and was comparable to that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway-initiated TG compared to 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 that a component of this contribution occurs in a FXII-independent manner.

Contact system and intrinsic pathway activation in patients with advanced pancreatic cancer: a prospective cohort study.

BACKGROUND: Despite high risk of venous thromboembolism (VTE) in patients with pancreatic cancer, there are little data on contact system activation in these patients. OBJECTIVES: To quantify contact system and intrinsic pathway activation and subsequent VTE risk in patients with pancreatic cancer. METHODS: Patients with advanced pancreatic cancer were compared with controls. Blood was drawn at baseline and patients were followed for 6 months. Complexes of proteases with their natural inhibitors, C1-esterase inhibitor (C1-INH), antithrombin (AT), or alpha-1 antitrypsin (α1at), were measured for complexes containing kallikrein (PKa:C1-INH), factor (F)XIIa (FXIIa:C1-INH), and FXIa (FXIa:C1-INH, FXIa:AT, FXIa:α1at). The association of cancer with complex levels was assessed in a linear regression model, adjusted for age, sex, and body mass index. In a competing risk regression model, we assessed associations between complex levels and VTE. RESULTS: One hundred nine patients with pancreatic cancer and 22 controls were included. The mean age was 66 years (SD, 8.4) in the cancer cohort and 52 years (SD, 10.1) in controls. In the cancer cohort, 18 (16.7%) patients developed VTE during follow-up. In the multivariable regression model, pancreatic cancer was associated with increased complexes of PKa:C1-INH (P < .001), FXIa:C1-INH (P < .001), and FXIa:AT (P < .001). High FXIa:α1at (subdistribution hazard ratio, 1.48 per log increase; 95% CI, 1.02-2.16) and FXIa:AT (subdistribution hazard ratio, 2.78 highest vs lower quartiles; 95% CI, 1.10-7.00) were associated with VTE. CONCLUSION: Complexes of proteases with their natural inhibitors were elevated in patients with cancer. These data suggest that the contact system and intrinsic pathway activation are increased in patients with pancreatic cancer.

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Absence of hyperfibrinolysis may explain lack of efficacy of tranexamic acid in hypoproliferative thrombocytopenia.

The American Trial Using Tranexamic Acid (TXA) in Thrombocytopenia (A-TREAT, NCT02578901) demonstrated no superiority of TXA over placebo in preventing World Health Organization (WHO) grade 2 or higher bleeding in patients with severe thrombocytopenia requiring supportive platelet transfusion following myeloablative therapy for hematologic disorders. In this ancillary study, we sought to determine whether this clinical outcome could be explained on the basis of correlative assays of fibrinolysis. Plasma was collected from A-TREAT participants (n = 115) before the initiation of study drug (baseline) and when TXA was at steady-state trough concentration (follow-up). Global fibrinolysis was measured by 3 assays: euglobulin clot lysis time (ECLT), plasmin generation (PG), and tissue-type plasminogen activator (tPA)-challenged clot lysis time (tPA-CLT). TXA was quantified in follow-up samples by tandem mass spectrometry. Baseline samples did not demonstrate fibrinolytic activation by ECLT or tPA-CLT. Furthermore, neither ECLT nor levels of plasminogen activator inhibitor-1, tPA, plasminogen, alpha2-antiplasmin, or plasmin-antiplasmin complexes were associated with a greater risk of WHO grade 2+ bleeding. TXA trough concentrations were highly variable (range, 0.7-10 µg/mL) and did not correlate with bleeding severity, despite the fact that plasma TXA levels correlated strongly with pharmacodynamic assessments by PG (Spearman r, -0.78) and tPA-CLT (r, 0.74). We conclude that (1) no evidence of fibrinolytic activation was observed in these patients with thrombocytopenia, (2) trough TXA concentrations varied significantly between patients receiving the same dosing schedule, and (3) tPA-CLT and PG correlated well with TXA drug levels.

Hypoxic storage of donor red cells preserves deformability after exposure to plasma from adults with sickle cell disease.

BACKGROUND: Red cell (RBC) transfusions are beneficial for patients with sickle cell disease (SCD), but ex vivo studies suggest that inflamed plasma from patients with SCD during crises may damage these RBCs, diminishing their potential efficacy. The hypoxic storage of RBCs may improve transfusion efficacy by minimizing the storage lesion. We tested the hypotheses that (1) The donor RBCs exposed to the plasma of patients in crisis would have lower deformability and higher hemolysis than those exposed to non-crisis plasma, and (2) hypoxic storage, compared to standard storage, of donor RBCs could preserve deformability and reduce hemolysis. STUDY DESIGN AND METHODS: 18 SCD plasma samples from patients who had severe acute-phase symptoms (A-plasma; n = 9) or were at a steady-state (S = plasma; n = 9) were incubated with 16 RBC samples from eight units that were stored either under conventional(CRBC) or hypoxic(HRBC) conditions. Hemolysis and microcapillary deformability assays of these RBCs were analyzed using linear mixed-effect models after each sample was incubated in patient plasma overnight at 37°C RESULTS: Relative deformability was 0.036 higher (p < 0.0001) in HRBC pairs compared to CRBC pairs regardless of plasma type. Mean donor RBC hemolysis was 0.33% higher after incubation with A-plasma compared to S-plasma either with HRBC or CRBC (p = 0.04). HRBCs incubated with steady-state patient plasma demonstrated the highest deformability and lowest hemolysis. CONCLUSION: Hypoxic storage significantly influenced RBC deformability. Patient condition significantly influenced post-incubation hemolysis. Together, HRBCs in steady-state plasma maximized donor red cell ex vivo function and survival.

Antithrombin-III mitigates thrombin-mediated endothelial cell contraction and sickle red blood cell adhesion in microscale flow.

Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin-III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient-derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin-activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule-1 (VCAM-1) mediated by thrombin. Our findings suggest that, by attenuating thrombin-mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.

Contact and intrinsic coagulation pathways are activated and associated with adverse clinical outcomes in COVID-19.

Coagulation activation is a prominent feature of severe acute respiratory syndrome coronavirus 2 (COVID-19) infection. Activation of the contact system and intrinsic pathway has increasingly been implicated in the prothrombotic state observed in both sterile and infectious inflammatory conditions. We therefore sought to assess activation of the contact system and intrinsic pathway in individuals with COVID-19 infection. Baseline plasma levels of protease:serpin complexes indicative of activation of the contact and intrinsic pathways were measured in samples from inpatients with COVID-19 and healthy individuals. Cleaved kininogen, a surrogate for bradykinin release, was measured by enzyme-linked immunosorbent assay, and extrinsic pathway activation was assessed by microvesicle tissue factor-mediated factor Xa (FXa; MVTF) generation. Samples were collected within 24 hours of COVID-19 diagnosis. Thirty patients with COVID-19 and 30 age- and sex-matched controls were enrolled. Contact system and intrinsic pathway activation in COVID-19 was demonstrated by increased plasma levels of FXIIa:C1 esterase inhibitor (C1), kallikrein:C1, FXIa:C1, FXIa:α1-antitrypsin, and FIXa:antithrombin (AT). MVTF levels were also increased in patients with COVID-19. Because FIXa:AT levels were associated with both contact/intrinsic pathway complexes and MVTF, activation of FIX likely occurs through both contact/intrinsic and extrinsic pathways. Among the protease:serpin complexes measured, FIXa:AT complexes were uniquely associated with clinical indices of disease severity, specifically total length of hospitalization, length of intensive care unit stay, and extent of lung computed tomography changes. We conclude that the contact/intrinsic pathway may contribute to the pathogenesis of the prothrombotic state in COVID-19. Larger prospective studies are required to confirm whether FIXa:AT complexes are a clinically useful biomarker of adverse clinical outcomes.

Euglobulin clot lysis time reveals a high frequency of fibrinolytic activation in trauma.

Activation of the fibrinolytic system plays a central role in the host response to trauma. There is significant heterogeneity in the degree of fibrinolysis activation at baseline that is usually assessed by whole blood thromboelastography (TEG). Few studies have focused on plasma markers of fibrinolysis that could add novel insights into the frequency and mechanisms of fibrinolytic activation in trauma. Global fibrinolysis in plasma was assessed using a modified euglobulin clot lysis time (ECLT) assay in 171 major trauma patients and compared to commonly assessed analytes of fibrinolysis. The median ECLT in trauma patients was significantly shorter at 8.5 h (IQR, 1.3-19.5) compared to 19.9 h (9.8-22.6) in healthy controls (p < 0.0001). ECLT values ≤2.5th percentile of the reference range were present in 83 (48.5%) of trauma patients, suggesting increased fibrinolytic activation. Shortened ECLT values were associated with elevated plasmin-antiplasmin (PAP) complexes and free tissue plasminogen activator (tPA) levels in plasma. Sixteen (9.2%) individuals met the primary outcome for massive transfusion, here defined as the critical administration threshold (CAT) of 3 units of packed red cells in any 60-minute period within the first 24 h. In a univariate screen, plasma biomarkers associated with CAT included D-dimer (p < 0.001), PAP (p < 0.05), free tPA (p < 0.05) and ECLT (p < 0.05). We conclude that fibrinolytic activation, measured by ECLT, is present in a high proportion of trauma patients at presentation. The shortened ECLT is partially driven by high tPA levels and is associated with high levels of circulating PAP complexes. Further studies are needed to determine whether ECLT is an independent predictor of trauma outcomes.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Tranexamic acid rapidly inhibits fibrinolysis, yet transiently enhances plasmin generation in vivo.

Tranexamic acid (TXA) is a lysine analogue that inhibits plasmin generation and has been used for decades as an antifibrinolytic agent to reduce bleeding. Recent reports have indicated that TXA can paradoxically promote plasmin generation. Blood was obtained from 41 cardiac surgical patients randomly assigned to TXA or placebo before start of surgery (preOP), at the end of surgery (EOS), then again on postoperative day 1 (POD-1) as well as POD-3. Plasma levels of tissue-type plasminogen activator (t-PA), urokinase (u-PA), the plasmin-antiplasmin (PAP) complex, as well as t-PA and u-PA-induced clot lysis assays were then determined. Clot lysis and PAP complex levels were also assessed in healthy volunteers before and at various time points after taking 1 g TXA orally. Surgery induced an increase in circulating t-PA, yet not u-PA at EOS. t-PA levels were unaffected by TXA; however, u-PA levels were significantly reduced in patients on POD-3. t-PA and u-PA-induced clot lysis were both inhibited in plasma from TXA-treated patients. In contrast, PAP complex formation, representing plasmin generation, was unexpectedly enhanced in the plasma of patients administered TXA at the EOS time point. In healthy volunteers, oral TXA effectively blocked fibrinolysis within 30 min and blockade was sustained for 8 h. However, TXA also increased PAP levels in volunteers 4 h after administration. Our findings demonstrate that TXA can actually augment PAP complex formation, consistent with an increase in plasmin generation in vivo despite the fact that it blocks fibrinolysis within 30 min. This may have unanticipated consequences in vivo.

Biomarker-enhanced VTE risk stratification in ambulatory patients with cancer.

INTRODUCTION: Risk assessment models are used to stratify cancer patients according to their underlying risk of VTE. The CATS score has been shown to enhance VTE risk stratification as compared to the modified Khorana score by incorporating d-dimer and soluble p-selectin measurements. Our aim was to evaluate the performance of the CATS score with respect to VTE risk stratification. MATERIALS AND METHODS: Analysis of a subset of the AVERT trial population for whom biomarker data was available. All patients included in the AVERT trial were at increased risk of VTE based on a modified Khorana score of ≥2. Patients were stratified according to the modified Khorana score and CATS score. Kaplan-Meier analysis was used to calculate the 6-month cumulative probabilities of VTE. RESULTS: A total of 466 patients were included in the analysis, 229 and 237 patients in the placebo and apixaban arms, respectively. The 6-month cumulative probability of VTE among patients with a modified Khorana score ≥ 3 was 13% [95% CI 7 to 23], whereas it was 20% [95% CI 11 to 35] for patients with a CATS score ≥ 4. The absolute risk reduction achieved with apixaban VTE prophylaxis among patients with modified Khorana ≥2, modified Khorana ≥3 and CATS ≥4 was -5.9% [-10.9 to -0.8], -5.8% [-16.0 to 4.5] and -10.1% [-22.9 to 2.6], respectively. Apixaban VTE prophylaxis among patients with increasing modified Khorana or CATS scores was not associated with an increased risk of bleeding events. CONCLUSIONS: The use of a CATS score of ≥4 to identify ambulatory cancer patients at very high risk of VTE could enhance the benefit/risk ratio achieved with apixaban VTE prophylaxis.

D-Dimer Enhances Risk-Targeted Thromboprophylaxis in Ambulatory Patients with Cancer.

BACKGROUND: Thromboprophylaxis for ambulatory patients with cancer is effective, although uncertainties remain on who should be targeted. Using D-dimer values from individuals enrolled to the AVERT trial, we sought to identify and validate a more efficient venous thromboembolism (VTE) risk threshold for thromboprophylaxis. MATERIALS AND METHODS: The AVERT trial compared thromboprophylaxis with apixaban with placebo among patients with cancer with a Khorana Risk Score ≥2. The D-dimer measured at randomization was used to calculate an individualized 6-month VTE risk using the validated CATScore. A modified intention-to-treat analysis was used to assess efficacy (VTE) and safety (major and overall bleeding) in the (a) complete cohort and (b) ≥8% and < 8% 6-month VTE risk thresholds. RESULTS: Five hundred seventy-four patients were randomized in the AVERT trial; 466 (81%) with baseline D-dimer were included in the study. Two hundred thirty-seven subjects received apixaban; 229 received placebo. In the complete cohort, there were 13 (5.5%) VTE events in the apixaban arm compared with 26 (11.4%) events in the placebo arm (adjusted hazard ratio [aHR] 0.49 [0.25-0.95], p < .05). Number needed to treat (NNT) to prevent one VTE = 17. Eighty-two (35%) and 72 (31%) patients in the apixaban and placebo arms, respectively, had a 6-month VTE risk ≥8%. In this subgroup, 7 (8.4%) VTE events occurred with apixaban and 19 (26.3%) events with placebo (aHR 0.33 [0.14-0.81], p < .05), NNT = 6. Individuals with a VTE risk <8% derived no benefit from apixaban thromboprophylaxis (aHR 0.89 [0.30-2.65), p = .84). Increased rates of overall bleeding were observed with apixaban in both the complete (aHR 2.11 [1.09-4.09], p < .05) and ≥ 8% predicted risk cohorts (aHR 2.87 [0.91-9.13], p = .07). CONCLUSION: A 6-month VTE risk threshold of ≥8% increases the efficiency of risk-targeted thromboprophylaxis in ambulatory patients with cancer. IMPLICATIONS FOR PRACTICE: Ambulatory patients with cancer receiving chemotherapy have an increased risk of venous thromboembolism (VTE). A Khorana Risk Score (KRS) ≥2 is currently the suggested threshold for thromboprophylaxis. Using baseline D-dimer values from individuals enrolled to the AVERT trial, this retrospective validation study identifies a 6-month VTE risk of ≥8% as a more efficient threshold for thromboprophylaxis. At this threshold, the number needed to treat to prevent one VTE is 6, compared with 17 when using a KRS ≥2. Conversely, individuals with a predicted risk of <8% derive no clinical benefit from thromboprophylaxis. Future prospective studies should validate this threshold for outpatient thromboprophylaxis.

Red blood cell adhesion to ICAM-1 is mediated by fibrinogen and is associated with right-to-left shunts in sickle cell disease.

Sickle cell disease (SCD), which afflicts 100 000 Americans, as well as millions worldwide, is associated with anemia, lifelong morbidity, and early mortality. Abnormal adhesion of sickle red blood cells (RBCs) to activated vascular endothelium may contribute acutely to the initiation of painful vaso-occlusive crises and chronically to endothelial damage in SCD. Sickle RBCs adhere to activated endothelium through several adhesion mechanisms. In this study, using whole blood from 17 people with heterozygous SCD (HbS variant) and 55 people with homozygous SCD (HbSS) analyzed in an in vitro microfluidic assay, we present evidence for the adhesion of sickle RBCs to immobilized recombinant intercellular adhesion molecule 1 (ICAM-1). We show that sickle RBC adhesion to ICAM-1 in vitro is associated with evidence of hemolysis in vivo, marked by elevated lactate dehydrogenase levels, reticulocytosis, and lower fetal hemoglobin levels. Further, RBC adhesion to ICAM-1 correlates with a history of intracardiac or intrapulmonary right-to-left shunts. Studies of potential ICAM-1 ligands on RBC membranes revealed that RBC-ICAM-1 interactions were mediated by fibrinogen bound to the RBC membrane. We describe, for the first time, RBC rolling behavior on ICAM-1 under high shear rates. Our results suggest that firm adhesion of sickle RBCs to ICAM-1 most likely occurs in postcapillary venules at low physiological shear rates, which is facilitated by initial rolling in high shear regions (eg, capillaries). Inhibition of RBC and ICAM-1 interactions may constitute a novel therapeutic target in SCD.

Biomarkers in cancer patients at risk for venous thromboembolism: data from the AVERT study.

BACKGROUND: The mechanisms surrounding cancer-associated venous thromboembolism (VTE) are not well characterized. AVERT, a randomized placebo controlled thromboprophylaxis study in ambulatory cancer patients, provides a unique opportunity to gain insights into thrombotic mechanism(s). METHODS: All available citrated platelet-free plasma samples collected at the point of randomization from individuals enrolled in the AVERT study were evaluated for the expression of D-dimer, soluble P-selectin (sP- selectin), active plasminogen activator inhibitor 1 (aPAI-1), clot lysis time (CLT) and activated factor XIa-C1 inhibitor complex (FXIa-C1). We compared the differential expression of sP-selectin, aPAI-1, CLT and FXIa-C1 among individual tumor types with normal controls. We evaluated the impact of disease type (hematologic versus solid organ malignancy) and stage (metastatic versus non-metastatic) on individual biomarker expression. RESULTS: We included 449 AVERT participants in this analysis. Baseline expression of the selected thrombosis biomarkers differed significantly by individual tumor type compared with normal controls. Levels of aPAI-1, CLT, FXIa-C1 and sP-selectin were significantly elevated in individuals with lymphoma compared to individuals with non-metastatic solid organ malignancies (p<0.05). Individuals with metastatic solid organ disease had elevated levels of D-dimer and sP-selectin compared to those with non-metastatic disease (p<0.05). CONCLUSION: Among a cohort of ambulatory patients at intermediate to high risk of VTE, these exploratory findings suggest that baseline activation of coagulation and fibrinolysis pathways vary significantly by tumor type and disease stage.

Protease: Serpin complexes to assess contact system and intrinsic pathway activation.

Mounting evidence suggests that a variety of disease states are pathophysiologically related to activation of the contact system in vivo. The plasma contact system is composed of a cascade of serine proteases initiated by surface activation of factor XII, which can then proceed through a procoagulant pathway by activating the intrinsic coagulation factor XI, or a proinflammatory pathway by activating prekallikrein. Serpins are the primary endogenous inhibitors of the contact system, which irreversibly inhibit their respective protease(s), forming a stable complex. We modified an existing assay strategy for detecting these complexes in plasma using ELISAs and determined the effect of preanalytical variation caused by anticoagulant selection and processing time. The assays were sensitive and specific to inherited deficiency of individual contact factors. We conclude that these assays are robust and represent a relatively simple approach to the assessment of contact factor activation in plasma samples.

Effect of chemotherapy and longitudinal analysis of circulating extracellular vesicle tissue factor activity in patients with pancreatic and colorectal cancer.

INTRODUCTION: We conducted a longitudinal study in patients with pancreatic and colorectal cancer. We determined the effect of chemotherapy on extracellular vesicle tissue factor (EVTF) activity and the association of plasma EVTF activity with venous thromboembolism (VTE) and survival. MATERIAL AND METHODS: We enrolled 13 patients with pancreatic and 22 patients with colorectal cancer. Plasma samples were collected during the 85-day study period. Patients were followed for 3 months after the study period. We recorded symptomatic VTE during the study period (3 months) or asymptomatic deep vein thrombosis detected by ultrasound at day 85. We measured EVTF activity before and after chemotherapy. RESULTS AND CONCLUSIONS: In the pancreatic cancer group, 2 patients had elevated levels of EVTF activity. One of these patients developed symptomatic VTE and died, and the second patient did not have a VTE but died. Chemotherapy decreased EVTF activity in 2 pancreatic patients with high levels. In the colorectal cancer group, 4 patients developed VTE, but EVTF activity was not elevated in any patient and no patient died. We observed a borderline significant correlation between EVTF activity and D-dimer in the patients with pancreatic but not colorectal cancer. In this small descriptive study, 2 patients with pancreatic cancer had an elevated level of EVTF activity. Both patients died during the study period, and one had a VTE. Chemotherapy decreased EVTF activity in these patients. In contrast, elevated levels of EVTF activity were not observed in patients with colorectal cancer with or without VTE.

Development and application of global assays of hyper- and hypofibrinolysis.

Numerous methods for evaluation of global fibrinolytic activity in whole blood or plasma have been proposed, with the majority based on tissue-type plasminogen activator (t-PA) addition to initiate fibrinolysis. We propose that such an approach is useful to reveal hypofibrinolysis, but t-PA concentrations should be kept to a minimum. In this paper, we describe a low-concentration t-PA plasma turbidity assay to evaluate several congenital factor deficiencies, including plasminogen activator inhibitor-1 (PAI-1) and plasminogen deficiency, as well as hemophilia A and B. In addition, we demonstrate a threshold dependency on endogenous PAI-1 levels. To assess endogenous hyperfibrinolysis, we suggest that assays that avoid t-PA addition are preferable, with assays based on euglobulin fractionation remaining a viable choice. We describe a euglobulin fraction clot lysis time (ECLT) assay with spectrophotometric readout and other modifications, and evaluate it as a tool to measure hyperfibrinolysis in inherited clotting factor deficiency states. We demonstrate that the ECLT is predominantly driven by residual amounts of PAI-1, t-PA, and α2-antiplasmin. These assays should be further evaluated for the detection of hypo- or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders.