Predictors and outcomes of acute pulmonary embolism in COVID-19; insights from US National COVID cohort collaborative.

OBJECTIVES: To investigate whether COVID-19 patients with pulmonary embolism had higher mortality and assess the utility of D-dimer in predicting acute pulmonary embolism. PATIENTS AND METHODS: Using the National Collaborative COVID-19 retrospective cohort, a cohort of hospitalized COVID-19 patients was studied to compare 90-day mortality and intubation outcomes in patients with and without pulmonary embolism in a multivariable cox regression analysis. The secondary measured outcomes in 1:4 propensity score-matched analysis included length of stay, chest pain incidence, heart rate, history of pulmonary embolism or DVT, and admission laboratory parameters. RESULTS: Among 31,500 hospitalized COVID-19 patients, 1117 (3.5%) patients were diagnosed with acute pulmonary embolism. Patients with acute pulmonary embolism were noted to have higher mortality (23.6% vs.12.8%; adjusted Hazard Ratio (aHR) = 1.36, 95% CI [1.20-1.55]), and intubation rates (17.6% vs. 9.3%, aHR = 1.38[1.18-1.61]). Pulmonary embolism patients had higher admission D-dimer FEU (Odds Ratio(OR) = 1.13; 95%CI [1.1-1.15]). As the D-dimer value increased, the specificity, positive predictive value, and accuracy of the test increased; however, sensitivity decreased (AUC 0.70). At cut-off D-dimer FEU 1.8 mcg/ml, the test had clinical utility (accuracy 70%) in predicting pulmonary embolism. Patients with acute pulmonary embolism had a higher incidence of chest pain and history of pulmonary embolism or deep vein thrombosis. CONCLUSIONS: Acute pulmonary embolism is associated with worse mortality and morbidity outcomes in COVID-19. We present D-dimer as a predictive risk tool in the form of a clinical calculator for the diagnosis of acute pulmonary embolism in COVID-19.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.

Blocking inhibition of prothrombinase by tissue factor pathway inhibitor alpha: a procoagulant property of heparins.

Unfractionated heparin (UFH) has procoagulant activity in antithrombin/heparin cofactor II (HCII)-depleted plasma. UFH prevents tissue factor pathway inhibitor alpha (TFPIα) from inhibiting the procoagulant enzyme complex, prothrombinase, providing a possible mechanism for its procoagulant activity. The procoagulant potential of UFH and various low molecular weight heparins (LMWHs) were characterized for TFPIα dependence, using thrombin generation assays performed with antithrombin/HCII-depleted plasma. UFH, the LMWHs enoxaparin and dalteparin, and the low anticoagulant LMWH 2-O, 3-O desulphated heparin (ODSH) all promoted thrombin generation, but fondaparinux did not, and this activity was blocked by a TFPIα antibody. UFH, enoxaparin, and dalteparin were anticoagulant in reactions containing 1-2% normal plasma. In prothrombinase activity assays, UFH, enoxaparin, dalteparin and ODSH blocked prothrombinase inhibition by TFPIα, while again fondaparinux did not. In both the plasma and purified assays, LMWHs displayed greater procoagulant potential than UFH, even when normalized to saccharide concentration. These biochemical data reveal that UFH and LMWHs, but not fondaparinux, block prothrombinase inhibition by TFPIα, thereby producing their paradoxical procoagulant activity observed in the absence of antithrombin/HCII. The findings may help to understand the complex pathophysiology and treatment of patients that are simultaneously bleeding and clotting, such as those with disseminated intravascular coagulation.

Biology of tissue factor pathway inhibitor.

Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIß, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIß have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5' untranslated region allows for translational regulation of TFPIß expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIß has the K1 and K2 domains attached to a glycosylphosphatidyl inositol-anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIß on the cell surface. TFPIα and TFPIß inhibit both TF-factor VIIa-dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIß may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation.

Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIß. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIß and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIß in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIß acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

Protein S is a cofactor for platelet and endothelial tissue factor pathway inhibitor-α but not for cell surface-associated tissue factor pathway inhibitor.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIß, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIß are undescribed. Thus, the physiological role and scope of the PS-TFPI system remain unclear. APPROACH AND RESULTS: Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIß was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIß or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. CONCLUSIONS: The PS-TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS-TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity.

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca(2+)-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC(50) = 0.3µM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

Caveolae optimize tissue factor-Factor VIIa inhibitory activity of cell-surface-associated tissue factor pathway inhibitor.

TFPI (tissue factor pathway inhibitor) is an anticoagulant protein that prevents intravascular coagulation through inhibition of fXa (Factor Xa) and the TF (tissue factor)-fVIIa (Factor VIIa) complex. Localization of TFPI within caveolae enhances its anticoagulant activity. To define further how caveolae contribute to TFPI anticoagulant activity, CHO (Chinese-hamster ovary) cells were co-transfected with TF and membrane-associated TFPI targeted to either caveolae [TFPI-GPI (TFPI-glycosylphosphatidylinositol anchor chimaera)] or to bulk plasma membrane [TFPI-TM (TFPI-transmembrane anchor chimaera)]. Stable clones had equal expression of surface TF and TFPI. TX-114 cellular lysis confirmed localization of TFPI-GPI to detergent-insoluble membrane fractions, whereas TFPI-TM localized to the aqueous phase. TFPI-GPI and TFPI-TM were equally effective direct inhibitors of fXa in amidolytic assays. However, TFPI-GPI was a significantly better inhibitor of TF-fVIIa than TFPI-TM, as measured in both amidolytic and plasma-clotting assays. Disrupting caveolae by removing membrane cholesterol from EA.hy926 cells, which make TFPIα, CHO cells transfected with TFPIß and HUVECs (human umbilical vein endothelial cells) did not affect their fXa inhibition, but significantly decreased their inhibition of TF-fVIIa. These studies confirm and quantify the enhanced anticoagulant activity of TFPI localized within caveolae, demonstrate that caveolae enhance the inhibitory activity of both TFPI isoforms and define the effect of caveolae as specifically enhancing the anti-TF activity of TFPI.

Novel strategies in antithrombotic therapy: targeting thrombosis while preserving hemostasis.

Antithrombotic therapy is a delicate balance between the benefits of preventing a thrombotic event and the risks of inducing a major bleed. Traditional approaches have included antiplatelet and anticoagulant medications, require careful dosing and monitoring, and all carry some risk of bleeding. In recent years, several new targets have been identified, both in the platelet and coagulation systems, which may mitigate this bleeding risk. In this review, we briefly describe the current state of antithrombotic therapy, and then present a detailed discussion of the new generation of drugs that are being developed to target more safely existing or newly identified pathways, alongside the strategies to reverse direct oral anticoagulants, showcasing the breadth of approaches. Combined, these exciting advances in antithrombotic therapy bring us closer than we have ever been to the "holy grail" of the field, a treatment that separates the hemostatic and thrombotic systems, preventing clots without any concurrent bleeding risk.

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation.

INTRODUCTION: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. MATERIALS AND METHODS: We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. RESULTS: In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. CONCLUSIONS: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.

Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation.

Systemic blood coagulation accompanies inflammation during severe infection like sepsis and COVID. We've previously established a link between pyroptosis, a vital defense mechanism against infection, and coagulopathy. During pyroptosis, the formation of gasdermin-D (GSDMD) pores on the plasma membrane leads to the release of tissue factor (TF)-positive microvesicles (MVs) that are procoagulant. Mice lacking GSDMD release fewer TF MVs. However, the specific mechanisms leading from activation of GSDMD to MV release remain unclear. Plasma membrane rupture (PMR) in pyroptosis was recently reported to be actively mediated by the transmembrane protein Ninjurin-1 (NINJ1). Here we show that NINJ1 promotes procoagulant MV release during pyroptosis. Haploinsuffciency or glycine inhibition of NINJ1 limited the release of procoagulant MVs and inflammatory cytokines and protected against blood coagulation and lethality triggered by bacterial flagellin. Our findings suggest a crucial role for NINJ1-dependent PMR in inflammasome-induced blood coagulation and inflammation.

Plasma tissue-type plasminogen activator is associated with lipoprotein(a) and clinical outcomes in hospitalized patients with COVID-19.

Background: Patients with COVID-19 have a higher risk of thrombosis and thromboembolism, but the underlying mechanism(s) remain to be fully elucidated. In patients with COVID-19, high lipoprotein(a) (Lp(a)) is positively associated with the risk of ischemic heart disease. Lp(a), composed of an apoB-containing particle and apolipoprotein(a) (apo(a)), inhibits the key fibrinolytic enzyme, tissue-type plasminogen activator (tPA). However, whether the higher Lp(a) associates with lower tPA activity, the longitudinal changes of these parameters in hospitalized patients with COVID-19, and their correlation with clinical outcomes are unknown. Objectives: To assess if Lp(a) associates with lower tPA activity in COVID-19 patients, and how in COVID-19 populations Lp(a) and tPA change post infection. Methods: Endogenous tPA enzymatic activity, tPA or Lp(a) concentration were measured in plasma from hospitalized patients with and without COVID-19. The association between plasma tPA and adverse clinical outcomes was assessed. Results: In hospitalized patients with COVID-19, we found lower tPA enzymatic activity and higher plasma Lp(a) than that in non-COVID-19 controls. During hospitalization, Lp(a) increased and tPA activity decreased, which associates with mortality. Among those who survived, Lp(a) decreased and tPA enzymatic activity increased during recovery. In patients with COVID-19, tPA activity is inversely correlated with tPA concentrations, thus, in another larger COVID-19 cohort, we utilized plasma tPA concentration as a surrogate to inversely reflect tPA activity. The tPA concentration was positively associated with death, disease severity, plasma inflammatory, and prothrombotic markers, and with length of hospitalization among those who were discharged. Conclusion: High Lp(a) concentration provides a possible explanation for low endogenous tPA enzymatic activity, and poor clinical outcomes in patients with COVID-19.

Dysregulation of Protein S in COVID-19.

Coronavirus Disease 2019 (COVID-19) has been widely associated with increased thrombotic risk, with many different proposed mechanisms. One such mechanism is acquired deficiency of protein S (PS), a plasma protein that regulates coagulation and inflammatory processes, including complement activation and efferocytosis. Acquired PS deficiency is common in patients with severe viral infections and has been reported in multiple studies of COVID-19. This deficiency may be caused by consumption, degradation, or clearance of the protein, by decreased synthesis, or by binding of PS to other plasma proteins, which block its anticoagulant activity. Here, we review the functions of PS, the evidence of acquired PS deficiency in COVID-19 patients, the potential mechanisms of PS deficiency, and the evidence that those mechanisms may be occurring in COVID-19.

Tissue factor release following traumatic brain injury drives thrombin generation.

Background: Traumatic brain injury (TBI) results in neurovascular damage that initiates intrinsic mechanisms of hypercoagulation, which can contribute to the development of life-threatening complications, such as coagulopathy and delayed thrombosis. Clinical studies have hypothesized that tissue factor (TF) induces hypercoagulability after TBI; however, none have directly shown this relationship. Objectives: In the current study, we took a stepwise approach to understand what factors are driving thrombin generation following experimental TBI. Methods: We employed the contusion-producing controlled cortical impact (CCI) model and the diffuse closed head injury (CHI) model to investigate these mechanisms as a function of injury severity and modality. Whole blood was collected at 6 hours and 24 hours after injury, and platelet-poor plasma was used to measure thrombin generation and extracellular vesicle (EV) TF. Results: We found that plasma thrombin generation, dependent on TF present in the plasma, was greater in CCI-injured animals compared to sham at both 6 hours (120.4 ± 36.9 vs 0.0 ± 0.0 nM*min endogenous thrombin potential) and 24 hours (131.0 ± 34.0 vs 32.1 ± 20.6 nM*min) after injury. This was accompanied by a significant increase in EV TF at 24 hours (328.6 ± 62.1 vs 167.7 ± 20.8 fM) after CCI. Further, EV TF is also increased at 6 hours (126.6 ± 17.1 vs 63.3 ± 14.4 fM) but not 24 hours following CHI. Conclusion: TF-mediated thrombin generation is time-dependent after injury and TF increases resolve earlier following CHI as compared to CCI. Taken together, these data support a TF-mediated pathway of thrombin generation after TBI and pinpoint TF as a major player in TBI-induced coagulopathy.

Total Plasma Protein S Is a Prothrombotic Marker in People Living With HIV.

BACKGROUND: HIV-1 infection is associated with multiple procoagulant changes and increased thrombotic risk. Possible mechanisms for this risk include heigthened expression of procoagulant tissue factor (TF) on circulating monocytes, extracellular vesicles, and viral particles and/or acquired deficiency of protein S (PS), a critical cofactor for the anticoagulant protein C (PC). PS deficiency occurs in up to 76% of people living with HIV-1 (PLWH). As increased ex vivo plasma thrombin generation is a strong predictor of mortality, we investigated whether PS and plasma TF are associated with plasma thrombin generation. METHODS: We analyzed plasma samples from 9 healthy controls, 17 PLWH on first diagnosis (naive), and 13 PLWH on antiretroviral therapy (ART). Plasma thrombin generation, total and free PS, PC, C4b-binding protein, and TF activity were measured. RESULTS: We determined that the plasma thrombin generation assay is insensitive to PS, because of a lack of PC activation, and developed a modified PS-sensitive assay. Total plasma PS was reduced in 58% of the naive and 38% of the ART-treated PLWH samples and correlated with increased thrombin generation in the modified assay. Conversely, plasma TF was not increased in our patient population, suggesting that it does not significantly contribute to ex vivo plasma thrombin generation. CONCLUSION: These data suggest that reduced total plasma PS contributes to the thrombotic risk associated with HIV-1 infection and can serve as a prothrombotic biomarker. In addition, our refined thrombin generation assay offers a more sensitive tool to assess the functional consequences of acquired PS deficiency in PLWH.