Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Protein disulfide isomerase uses thrombin-antithrombin complex as a template to bind its target protein and alter the blood coagulation rates.

During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.

Platelet Function Decreases with Increasing Severity of Liver Cirrhosis and Portal Hypertension-A Prospective Study.

BACKGROUND: Cirrhotic patients display an increased risk for both bleeding and thrombosis. We investigated platelet activation across Child-Pugh stages (CPSs) and portal hypertension (PH) severity. MATERIAL AND METHODS: A total of 110 cirrhotic patients were prospectively included. CPS and hepatic venous pressure gradient (HVPG) were determined. Platelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa were measured by flow cytometry before/after stimulation with protease-activated receptor (PAR)-1 (thrombin receptor activating peptide, TRAP) and PAR-4 (AYPGKF) agonists, epinephrine, and lipopolysaccharide (LPS). RESULTS: Platelet count was similar across CPS but lower with increasing PH severity. Expression of P-selectin and activated GPIIb/IIIa in response to TRAP and AYPGKF was significantly reduced in platelets of CPS-B/C versus CPS-A patients (all p < 0.05). Platelet P-selectin expression upon epinephrine and LPS stimulation was reduced in CPS-C patients, while activated GPIIb/IIIa in response to these agonists was lower in CPS-B/C (all p < 0.05). Regarding PH severity, P-selectin and activated GPIIb/IIIa in response to AYPGKF were lower in HVPG ≥20 mmHg patients (both p < 0.001 vs. HVPG < 10 mmHg). Similarly, activated GPIIb/IIIa was lower in HVPG ≥20 mmHg patients after TRAP stimulation (p < 0.01 vs. HVPG < 10 mmHg). The lower platelet surface expression of P-selectin and activated GPIIb/IIIa upon stimulation of thrombin receptors (PAR-1/PAR-4) in CPS-B/C and HVPG ≥20 mmHg patients was paralleled by reduced antithrombin-III levels in those patients (all p < 0.05). Overall, PAR-1- and PAR-4-mediated platelet activation correlated with antithrombin-III levels (p < 0.001). CONCLUSION: Platelet responsiveness decreases with increasing severity of liver cirrhosis and PH but is potentially counterbalanced by lower antithrombin-III levels.

The Interaction of Factor Xa and IXa with Non-Activated Antithrombin in Michaelis Complex: Insights from Enhanced-Sampling Molecular Dynamics Simulations.

The interaction between coagulation factors Xa and IXa and the activated state of their inhibitor, antithrombin (AT),have been investigated using X-ray diffraction studies. However, only mutagenesis data are available for non-activated AT. Our aim was to propose a model based on docking and advanced-sampling molecular dynamics simulations that can reveal the conformational behavior of the systems when AT is not binding a pentasaccharide. We built the initial structure for non-activated AT-FXa and AT-FIXa complexes using HADDOCK 2.4. The conformational behavior was studied using Gaussian accelerated molecular dynamics simulations. In addition to the docked complexes, two systems based on the X-ray structures were also simulated, with and without the ligand. The simulations revealed large variability in conformation for both factors. In the docking-based complex of AT-FIXa, conformations with stable Arg150-AT interactions can exist for longer time periods but the system also has a higher tendency for reaching states with very limited interaction with the "exosite" of AT. By comparing simulations with or without the pentasaccharide, we were able to gain insights into the effects of conformational activation on the Michaelis complexes. RMSF analysis and correlation calculations for the alpha-carbon atoms revealed important details of the allosteric mechanisms. Our simulations provide atomistic models for better understanding the conformational activation mechanism of AT against its target factors.

Characterization of antithrombin isoforms and latent forms by ion exchange chromatography coupled to mass spectrometry.

Antithrombin is a key protein of the coagulation system belonging to the serine protease inhibitor family. Antithrombin preparations are used as a therapeutic treatment for patients with decreased antithrombin activity. Elucidating the structural features of this protein is an important part of the control strategy to assure a high quality. This study presents an ion exchange chromatographic method coupled to mass spectrometry capable of characterizing antithrombin post-translational modifications such as N-glycosylation, phosphorylation or deamidation. Furthermore, the method was successfully used to evidence irreversible/inactive conformers of antithrombin which are commonly observed for serine protease inhibitors and referred to as latent forms.

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin.

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.

Full-length antithrombin frameshift variant with aberrant C-terminus causes endoplasmic reticulum retention with a dominant-negative effect.

Antithrombin, a major endogenous anticoagulant, is a serine protease inhibitor (serpin). We characterized the biological and clinical impact of variants involving C-terminal antithrombin. We performed comprehensive molecular, cellular, and clinical characterization of patients with C-terminal antithrombin variants from a cohort of 444 unrelated individuals with confirmed antithrombin deficiency. We identified 17 patients carrying 12 C-terminal variants, 5 of whom had the p.Arg445Serfs*17 deletion. Five missense variants caused qualitative deficiency, and 7, including 4 insertion-deletion variants, induced severe quantitative deficiency, particularly p.Arg445Serfs*17 (antithrombin <40%). This +1 frameshift variant had a molecular size similar to that of WT antithrombin but possessed a different C-terminus. Morphologic and cotransfection experiments showed that recombinant p.Arg445Serfs*17 was retained at the endoplasmic reticulum and had a dominant-negative effect on WT antithrombin. Characterization of different 1+ frameshift, aberrant C-terminal variants revealed that protein secretion was determined by frameshift site. The introduction of Pro441 in the aberrant C-terminus, shared by 5 efficiently secreted variants, partially rescued p.Arg445Serfs*17 secretion. C-terminal antithrombin mutants have notable heterogeneity, related to variant type and localization. Aberrant C-terminal variants caused by 1+ frameshift, with similar size as WT antithrombin, may be secreted or not, depending on frameshift site. The severe clinical phenotypes of these genetic changes are consistent with their dominant-negative effects.

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.

Implications of Heparanase on Heparin Synthesis and Metabolism in Mast Cells.

Heparin is a polysaccharide expressed in animal connective tissue-type mast cells. Owing to the special pentasaccharide sequence, heparin specifically binds to antithrombin (AT) and increases the inhibitory activity of AT towards coagulation enzymes. Heparin isolated from porcine intestinal mucosa has an average molecular weight of 15 kDa, while heparins recovered from rat skin and the peritoneal cavity were 60-100 kDa and can be fragmented by the endo-glucuronidase heparanase in vitro. In this study, we have examined heparin isolated from in vitro matured fetal skin mast cells (FSMC) and peritoneal cavity mast cells (PCMC) collected from wildtype (WT), heparanase knockout (Hpa-KO), and heparanase overexpressing (Hpa-tg) mice. The metabolically 35S-labeled heparin products from the mast cells of WT, Hpa-KO, and Hpa-tg mice were compared and analyzed for molecular size and AT-binding activity. The results show that PCMC produced heparins with a size similar to heparin from porcine intestinal mast cells, whilst FSMC produced much longer chains. As expected, heparanase overexpression resulted in the generation of smaller fragments in both cell types, while heparins recovered from heparanase knockout cells were slightly longer than heparin from WT cells. Unexpectedly, we found that heparanase expression affected the production of total glycosaminoglycans (GAGs) and the proportion between heparin and other GAGs but essentially had no effect on heparin catabolism.

Experiences in Routine Genetic Analysis of Hereditary Hemorrhagic, Thrombotic, and Platelet Disorders.

Hemostasis is a complex and tightly regulated system that attempts to maintain a homeostatic balance to permit normal blood flow, without bleeding or thrombosis. Hemostasis reflects the subtle balance between procoagulant and anticoagulant factors in the pathways of primary hemostasis, secondary hemostasis, and fibrinolysis. The major components in this interplay include the vascular endothelium, platelets, coagulation factors, and fibrinolytic factors. After vessel wall injury, the subendothelium is exposed to the blood stream, followed by rapid activation of platelets via collagen binding and von Willebrand factor-mediated platelet adhesion to the damaged vessel wall through platelet glycoprotein receptor Ib/IX/V. Activated platelets change their shape, release bioactive molecules from their granules, and expose negatively charged phospholipids on their surface. For a proper function of this process, an adequate number of functional platelets are required. Subsequently, a rapid generation of sufficient amounts of thrombin begins; followed by activation of the coagulation system and its coagulation factors (secondary hemostasis), generating fibrin that consolidates the platelet plug. To maintain equilibrium between coagulation and anticoagulation, the naturally occurring anticoagulants such as protein C, protein S, and antithrombin keep this process in balance. Deficiencies (inherited or acquired) at any level of this fine-tuned system result in pathologic bleedings or increased hypercoagulability states leading to thrombosis. This review will focus on genetic diagnosis of inherited bleeding, thrombotic, and platelet disorders, discussing strengths and limitations of existing diagnostic settings and genetic tools and highlight some important considerations necessary for clinical application.

Synthesis and evaluation of peptidic thrombin inhibitors bearing acid-stable sulfotyrosine analogues.

Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we report the synthesis of two sulfonate analogues and their incorporation into two thrombin-inhibiting sulfopeptides. The effective mimicry of these sulfonate analogues for native sulfotyrosine was validated in the context of their thrombin inhibitory activity and binding mode, as determined by X-ray crystallography.

Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability.

Hemophilia A and B are congenital bleeding disorders caused by a deficiency in pro-coagulant factor VIII or IX that is treated by downregulation of antithrombin. However, the molecular mechanisms that regulate antithrombin expression remain poorly understood. Here, we identified Cullin 2 and USP2 (ubiquitin-specific peptidase-2) as novel regulators of antithrombin expression that act by modulating antithrombin ubiquitination. Inhibition of the proteasome caused accumulation of antithrombin and its ubiquitinated forms in HepG2 and SMMC7721 cells. Notably, inhibition of neddylation with MLN4924 suppressed both ubiquitination and degradation of antithrombin, which is recapitulated by silencing of the neddylation enzymes, NAE1, UBA3, and UBE2M, with small interfering RNA (siRNA). We identified Cullin 2 as the interaction partner of antithrombin, and siRNA-mediated Cullin 2 knockdown reduced antithrombin ubiquitination and increased antithrombin protein. We further found that USP2 interacted with antithrombin and regulated antithrombin expression, showing that overexpression of USP2 inhibits the ubiquitination and proteasomal clearance of antithrombin, whereas pharmacological inhibition or siRNA-mediated knockdown of USP2 downregulates antithrombin. Collectively, these results suggest that Cullin 2 E3 ubiquitin ligase and USP2 coordinately regulate antithrombin ubiquitination and degradation. Thus, targeting Cullin 2 and USP2 could be a potential strategy for treatment of hemophilia.

Paramount Importance of Core Conformational Changes for Heparin Allosteric Activation of Antithrombin.

Antithrombin is unique among serpin family protein protease inhibitors with respect to the major reactive center loop (RCL) and core conformational changes that mediate allosteric activation of its anticoagulant function by heparin. A critical role for expulsion of the RCL hinge from a native stabilizing interaction with the hydrophobic core in the activation mechanism has been proposed from reports that antithrombin variants that block this change through engineered disulfide bonds block activation. However, the sufficiency of core conformational changes for activation without expulsion of the RCL from the core is suggested by variants that are activated without the need for heparin and retain the native RCL-core interaction. To resolve these apparently conflicting findings, we engineered variants in which disulfides designed to block the RCL conformational change were combined with constitutively activating mutations. Our findings demonstrate that while a reversible constitutive activation can be engineered in variants that retain the native RCL-core interaction, engineered disulfides that lock the RCL native conformation can also block heparin allosteric activation. Such findings support a three-state allosteric activation model in which constitutive activating mutations stabilize an intermediate-activated state wherein core conformational changes and a major activation have occurred without the release of the RCL from the core but with a necessary repositioning of the RCL to allow productive engagement with an exosite. Rigid disulfide bonds that lock the RCL native conformation block heparin activation by preventing both RCL repositioning in the intermediate-activated state and the release of the RCL from the core in the fully activated state.

Functional Characterization of Antithrombin Mutations by Monitoring of Thrombin Inhibition Kinetics.

Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.

De novo transcriptome reveals blood coagulation/antithrombin factors and infection mechanisms in Angiostrongylus cantonensis adult worms.

Angiostrongylus cantonensis is the main aetiological agent of eosinophilic meningoencephalitis in humans. Several outbreaks have been documented around the world, cementing its status as an emerging global public health concern. As a result, new strategies for the diagnosis, prophylaxis and treatment of cerebral angiostrongyliasis are urgently needed. In this study, we report on the de novo assembly of the A. cantonensis transcriptome, its full functional annotation and a reconstruction of complete metabolic pathways. All results are available at AngiostrongylusDB (http://angiostrongylus.lad.pucrs.br/admin/welcome). The aim of this study was to identify the active genes and metabolic pathways involved in the mechanisms of infection and survival inside Rattus norvegicus. Among 389 metabolic mapped pathways, the blood coagulation/antithrombin pathways of heparan sulphate/heparin are highlighted. Moreover, we identified genes codified to GP63 (leishmanolysin), CALR (calreticulin), ACE (peptidyl-dipeptidase A), myoglobin and vWD (von Willebrand factor type D domain protein) involved in the infection invasion and survival of the parasite. The large dataset of functional annotations provided and the full-length transcripts identified in this research may facilitate future functional genomics studies and provides a basis for the development of new techniques for the diagnosis, prevention and treatment of cerebral angiostrongyliasis.

Comprehensive review of the impact of direct oral anticoagulants on thrombophilia diagnostic tests: Practical recommendations for the laboratory.

There is a laboratory and clinical need to know the impact of direct oral anticoagulants (DOACs) on diagnostic tests to avoid misinterpretation of results. Although the regulatory labelling documents provide some information about the influences of each DOAC on diagnostic tests, these are usually limited to some of the most common tests and no head to head comparison is available. In this paper, we report the impact of DOACs on several thrombophilia tests, including assessment of antithrombin, protein S and protein C activity assays, detection of activated protein C resistance and assays used for lupus anticoagulant. Results are compared and discussed with data obtained from literature. The final goal of this comprehensive review is to provide practical recommendations for laboratories to avoid misdiagnosis due to oral direct factor Xa (FXa) or IIa (FIIa) inhibitors. Overall, oral direct FXa (apixaban, betrixaban, edoxaban and rivaroxaban) and FIIa (dabigatran) antagonists may affect clot-based thrombophilia diagnostic tests resulting in false-positive or false-negative results. An effect on FIIa-based thrombophilia diagnostic tests is observed with dabigatran but not with anti-FXa DOACs and conversely for FXa-based thrombophilia diagnostic tests. No impact was observed with antigenic/chromogenic methods for the assessment of protein S and C activity. In conclusion, interpretation of thrombophilia diagnostic tests results should be done with caution in patients on DOACs. The use of a device/chemical compound able to remove or antagonize the effect of DOACs or the development of new diagnostic tests insensitive to DOACs should be considered to minimize the risk of false results.

A quantitative method to detect non-antithrombin-binding 3-O-sulfated units in heparan sulfate.

Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of 13 units was prepared. Using reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system, the separation conditions were optimized to quantify the 13 units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units and establishes a new approach to explore the physiological functions of 3-O-sulfate.

Antithrombin Deficiency in Trauma and Surgical Critical Care.

Antithrombin deficiency (ATD) was described in 1965 by Olav Egeberg as the first known inherited form of thrombophilia. Today, it is understood that ATDs can be congenital or acquired, leading to qualitative, quantitative, or mixed abnormalities in antithrombin (AT). All ATDs ultimately hinder AT's ability to serve as an endogenous anticoagulant and antiinflammatory agent. As a result, ATD patients possess higher risk for thromboembolism and can develop recurrent venous and arterial thromboses. Because heparin relies on AT to augment its physiologic function, patients with ATD often exhibit profound heparin resistance. Although rare as a genetic disorder, acquired forms of ATD are seen with surprising frequency in critically ill patients. This review discusses ATD in the context of surgical critical care with specific relevance to trauma, thermal burns, cardiothoracic surgery, and sepsis.

Degeneracy of the Antithrombin Binding Sequence in Heparin: 2-O-Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid.

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this "canonical" GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1 C4 -2 S0 equilibrium in the unbound saccharide, shift to full 2 S0 and full 1 C4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.

Factor VIII and Functional Protein C Activity in Critically Ill Patients With Coronavirus Disease 2019: A Case Series.

Critically ill patients with coronavirus disease 2019 (COVID-19) have been observed to be hypercoagulable, but the mechanisms for this remain poorly described. Factor VIII is a procoagulant factor that increases during inflammation and is cleaved by activated protein C. To our knowledge, there is only 1 prior study of factor VIII and functional protein C activity in critically ill patients with COVID-19. Here, we present a case series of 10 critically ill patients with COVID-19 who had severe elevations in factor VIII activity and low normal functional protein C activity, which may have contributed to hypercoagulability.