Past meets present: Reviving 80-year-old Canadian dried serum from World War II and its significance in advancing modern freeze-dried plasma for prehospital management of haemorrhage.

During World War II, Charles H. Best utilized Charles R. Drew's plasma isolation and drying technique to lead Canada's initiative to provide dried serum as a means of primary resuscitation for British casualties on the frontlines. Serum was likely utilized over plasma for its volume expansion properties without the risk of clotting during prolonged storage. We reconstituted dried serum from 1943 and discovered intact albumin, as well as anti-thrombin, plasminogen, protein C and protein S activity. Proteomic analysis identified 71 proteins, most prominent being albumin, and positive for hepatitis B by serological testing. Transmission of blood-borne diseases ended the programme, until modern advances in testing and pathogen reduction revived this technology. We tested the latest iteration of Canadian freeze-dried plasma (FDP), which was stored for 4 years, and demonstrated that its clotting capacity remained equivalent to fresh frozen plasma. We recommend that FDP is a strong alternative to contemporary prehospital resuscitation fluids (e.g. normal saline/lactated Ringer's) in managing prehospital haemorrhage where whole blood is unavailable.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13.

Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1' substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF.

Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII.

Binding to the von Willebrand factor (VWF) D'D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D'D3 alone and in complex with FVIII. The D'D3 dimer ([D'D3]2) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D'. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D' domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. Modeling the FVIII structure into the three-dimensional EM reconstructions of [D'D3]2-FVIII ternary and quaternary complexes indicates conformational rearrangements of the FVIII C domains compared with their disposition in the unbound state. These results illustrate the cooperative plasticity between VWF and FVIII that coordinate their high-affinity interaction.

A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice.

Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D'D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf(-/-) mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D'D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf(-/-) mice. Although the VWF D'D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D'D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D'D3 and VWF D'D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D'D3/D'D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding.

Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function.

Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.

Von Willebrand factor: form for function.

The mechanisms by which von Willebrand factor (VWF) achieves hemostasis lie in its structure. Whereas low-molecular-weight forms have diminished hemostatic potential, ultralarge VWF (ULVWF) in excess is potentially thrombogenic. VWF comprises many subunits, which themselves comprise many repeated domains/assemblies possessing characteristic function(s). Organization of these domains/assemblies into a multimeric structure effectively links and replicates these functions. Each domain/assembly influences the synthesis, assembly, secretion, or hemostatic potential of plasma VWF. The C-terminal CT/CK domain mediates dimerization of VWF subunits in the endoplasmic reticulum, while the N-terminal D1D2 assemblies catalyzes disulfide binding between juxtaposed D3 assemblies in the trans-Golgi, creating multimers. The pH-sensitive domains (A2-CT/CK) allow ULVWF multimers to orderly pack into tubules that unravel upon secretion into the circulation. Hemodynamic forces regulate the conformation of the A2 domain and thus, its accessibility to proteolytic enzyme(s) that regulate VWF's hemostatic potential. Binding to the VWF D'D3 assemblies stabilizes coagulation factor VIII. The VWF A1 and A3 domains facilitate platelet capture onto exposed collagen(s) at sites of vascular injury. Our deeper understanding of VWF provided through the recent growth in VWF structure-function studies may potentially guide novel therapeutics for clotting or bleeding disorders.

Development of tag-free photoprobes for studies aimed at identifying the target of novel Group A Streptococcus antivirulence agents.

We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure-activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS-PAGE gels. This stepwise, 'tag-free' approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification.

A specific fluorescence resonance energy quenching-based biosensor for measuring thrombin activity in whole blood.

BACKGROUND: At sites of vessel injury, thrombin acts as the central mediator of coagulation by catalyzing fibrin clot formation and platelet activation. Thrombin generation is most frequently measured in plasma samples using small-molecule substrates; however, these have low specificity for thrombin and limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells and require anticoagulation and the addition of supraphysiological concentrations of calcium. OBJECTIVES: To overcome these limitations, we designed and characterized a fluorescence resonance energy quenching-based thrombin sensor (FTS) protein. METHODS: The fluorescence resonance energy quenching pair of mAmetrine and tTomato, separated by a thrombin recognition sequence, was developed. The protein was expressed using Escherichia coli, and purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cleavage of FTS was monitored by fluorescence using excitation at 406 nm and emission at 526 nm and 581 nm. RESULTS: Compared with small-molecule substrates, the FTS demonstrated high specificity for thrombin; it is not cleaved by thrombin or inhibited by α2-macroglobulin and interacts with thrombin's anion-binding exosite I. The FTS can effectively measure thrombin generation in plasma and in finger-prick whole blood, which allows it to be developed into a point-of-care test of thrombin generation. The FTS does not inhibit standard thrombin-generation assays. Lastly, FTS-based thrombin generation in nonanticoagulated finger-prick blood was delayed but enhanced compared with that in citrated plasma. CONCLUSION: The FTS will broaden our understanding of thrombin generation in ways that are not attainable with current methods.

REVIEWING THE DYSREGULATION OF ADAMTS13 AND VWF IN SEPSIS.

ABSTRACT: Sepsis is defined as a life-threatening organ dysfunction caused by excessive host response to infection, and represents the most common cause of in-hospital deaths. Sepsis accounts for 30% of all critically ill patients in the intensive care unit (ICU), and has a global mortality rate of 20%. Activation of blood coagulation during sepsis and septic shock can lead to disseminated intravascular coagulation, which is characterized by microvascular thrombosis. Von Willebrand factor (VWF) and ADAMTS13 are two important regulators of blood coagulation that may be important links between sepsis and mortality in the ICU. Herein we review our current understanding of VWF and ADAMTS13 in sepsis and other critical illnesses and discuss their contribution to disease pathophysiology, their use as markers of severe illness, and potential targets for new therapeutic development.

Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

Impact of age on the host response to sepsis in a murine model of fecal-induced peritonitis.

INTRODUCTION: Despite older adults being more vulnerable to sepsis, most preclinical research on sepsis has been conducted using young animals. This results in decreased scientific validity since age is an independent predictor of poor outcome. In this study, we explored the impact of aging on the host response to sepsis using the fecal-induced peritonitis (FIP) model developed by the National Preclinical Sepsis Platform (NPSP). METHODS: C57BL/6 mice (3 or 12 months old) were injected intraperitoneally with rat fecal slurry (0.75 mg/g) or a control vehicle. To investigate the early stage of sepsis, mice were culled at 4 h, 8 h, or 12 h to investigate disease severity, immunothrombosis biomarkers, and organ injury. Mice received buprenorphine at 4 h post-FIP. A separate cohort of FIP mice were studied for 72 h (with buprenorphine given at 4 h, 12 h, and then every 12 h post-FIP and antibiotics/fluids starting at 12 h post-FIP). Organs were harvested, plasma levels of Interleukin (IL)-6, IL-10, monocyte chemoattract protein (MCP-1)/CCL2, thrombin-antithrombin (TAT) complexes, cell-free DNA (CFDNA), and ADAMTS13 activity were quantified, and bacterial loads were measured. RESULTS: In the 12 h time course study, aged FIP mice demonstrated increased inflammation and injury to the lungs compared to young FIP mice. In the 72 h study, aged FIP mice exhibited a higher mortality rate (89%) compared to young FIP mice (42%) (p < 0.001). Aged FIP non-survivors also exhibited a trend towards elevated IL-6, TAT, CFDNA, CCL2, and decreased IL-10, and impaired bacterial clearance compared to young FIP non-survivors. CONCLUSION: To our knowledge, this is the first study to investigate the impact of age on survival using the FIP model of sepsis. Our model includes clinically-relevant supportive therapies and inclusion of both sexes. The higher mortality rate in aged mice may reflect increased inflammation and worsened organ injury in the early stage of sepsis. We also observed trends in impaired bacterial clearance, increase in IL-6, TAT, CFDNA, CCL2, and decreased IL-10 and ADAMTS13 activity in aged septic non-survivors compared to young septic non-survivors. Our aging model may help to increase the scientific validity of preclinical research and may be useful for identifying mechanisms of age-related susceptibility to sepsis as well as age-specific treatment strategies.

Metalloprotease domain latency protects ADAMTS13 against broad-spectrum inhibitors of metalloproteases while maintaining activity toward VWF.

BACKGROUND: ADAMTS13 is a circulating metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner. ADAMTS13 is secreted as an active protease but has a long half-life, suggesting that it is resistant to circulating protease inhibitors. These zymogen-like properties indicate that ADAMTS13 exists as a latent protease that is activated by its substrate. OBJECTIVES: To investigate the mechanism of ADAMTS13 latency and resistance to metalloprotease inhibitors. METHODS: Probe the active site of ADAMTS13 and variants using alpha-2 macroglobulin (A2M), tissue inhibitors of metalloproteases (TIMPs), and Marimastat. RESULTS: ADAMTS13 and C-terminal deletion mutants are not inhibited by A2M, TIMPs, or Marimastat, but cleave FRETS-VWF73, suggesting that the metalloprotease domain is latent in the absence of substrate. Within the metalloprotease domain, mutating the gatekeeper triad (R193, D217, D252) or substituting the calcium-binding (R180-R193) or the variable (G236-S263) loops with corresponding features from ADAMTS5 did not sensitize MDTCS to inhibition. However, substituting the calcium-binding loop and an extended variable loop (G236-S263) corresponding to the S1-S1' pockets with those from ADAMTS5, resulted in MDTCS-GVC5 inhibition by Marimastat, but not by A2M or TIMP3. Substituting the MD domains of ADAMTS5 into full-length ADAMTS13 resulted in a 50-fold reduction in activity compared with the substitution into MDTCS. However, both chimeras were susceptible to inhibition, suggesting that the closed conformation does not contribute to the latency of the metalloprotease domain. CONCLUSION: The metalloprotease domain protects ADAMTS13 from inhibitors and exists in a latent state that is partially maintained by loops flanking the S1 and S1' specificity pockets.

Mechanisms of ADAMTS13 regulation.

Recombinant ADAMTS13 is currently undergoing clinical trials as a treatment for hereditary thrombotic thrombocytopenic purpura, a lethal microvascular condition resulting from ADAMTS13 deficiency. Preclinical studies have also demonstrated its efficacy in treating arterial thrombosis and inflammation without causing bleeding, suggesting that recombinant ADAMTS13 may have broad applicability as an antithrombotic agent. Despite this progress, we currently do not understand the mechanisms that regulate ADAMTS13 activity in vivo. ADAMTS13 evades canonical means of protease regulation because it is secreted as an active enzyme and has a long half-life in circulation, suggesting that it is not inhibited by natural protease inhibitors. Although shear can spatially and temporally activate von Willebrand factor to capture circulating platelets, it is also required for cleavage by ADAMTS13. Therefore, spatial and temporal regulation of ADAMTS13 activity may be required to stabilize von Willebrand factor-platelet strings at sites of vascular injury. This review outlines potential mechanisms that regulate ADAMTS13 in vivo including shear-dependency, local inactivation, and biochemical and structural regulation of substrate binding. Recently published structural data of ADAMTS13 is discussed, which may help to generate novel hypotheses for future research.

Identification of the histidine-rich glycoprotein domains responsible for contact pathway inhibition.

BACKGROUND: Previously, we showed that histidine-rich glycoprotein (HRG) binds factor (F) XIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Therefore, HRG downregulates the contact pathway in vitro and in vivo. OBJECTIVE: To identify the domains on HRG responsible for contact pathway inhibition. METHODS: Recombinant HRG domain constructs (N-terminal [N1, N2, and N1N2], proline-rich regions, histidine-rich region [HRR], and C-terminal) were expressed and purified. The affinities of plasma-derived HRG, HRG domain constructs, and synthetic HRR peptides for FXII, FXIIa, ß-FXIIa, and polyphosphate (polyP) were determined using surface plasmon resonance, and their effects on polyP-induced FXII autoactivation, FXIIa-mediated activation of FXI and prekallikrein, the activated partial thromboplastin time (APTT), and thrombin generation were examined. RESULTS: HRG and HRG domain constructs bind FXIIa, but not FXII or ß-FXII. HRR, N1, and N1N2 bind FXIIa with affinities comparable with that of HRG, whereas the remaining domains bind with lower affinity. Synthetic HRR peptides bind FXIIa and polyP with high affinity. HRG and HRR significantly inhibit FXII autoactivation and prolong the APTT. Like HRG, synthetic HRR peptides inhibit FXII autoactivation, attenuate FXIIa-mediated activation of prekallikrein and FXI, prolong the APTT, and attenuate thrombin generation. CONCLUSION: The interaction of HRG with FXIIa and polyP is predominantly mediated by the HRR domain. Like intact HRG, HRR downregulates the contact pathway and contributes to HRG-mediated down regulation of coagulation.

Tissue factor and thrombosis models.

Mouse models of thrombosis have extended our understanding of the role of tissue factor (TF) in thrombogenesis. Because tissue factor deficiency is embryonic lethal in mice, inventive genetic models are required to probe the role of TF in thrombosis. TF is expressed by different cell types, including vascular smooth muscle cells, cardiomyocytes, fibroblasts, and monocytes. Platelets and endothelial cells also express TF under certain conditions, but the importance of this TF remains controversial. Animal models are commonly used to evaluate the contribution of TF from each cell type to thrombogenesis. Although a variety of well-established injury techniques are used to induce thrombosis, it is likely that the sources of TF that drive thrombosis are model dependent. Therefore, rigorous controls are needed before thrombogenesis can be attributed to TF from a particular cell type. This review summarizes data from mouse models that have attempted to delineate the role of TF in thrombus formation in response to various types of vascular injury. We have consolidated this information to generate unifying concepts that require testing in future studies.

Characterization of ADAMTS13 and von Willebrand factor levels in septic and non-septic ICU patients.

Sepsis is a life-threatening disease characterized by excessive host response to infection that can lead to activation of the coagulation system. Von Willebrand Factor (VWF) and ADAMTS13 are important regulators of hemostasis and their dysregulation during sepsis progression is not well understood. Herein we characterize ADAMTS13 and VWF in septic and non-septic patients. ADAMTS13 activity, ADAMTS13 antigen, VWF antigen, myeloperoxidase, and protein C, were measured in plasma collected from 40 septic patients (20 non-survivors and 20 survivors) and 40 non-septic patients on the first and last day of their ICU stay. ADAMTS13 activity and ADAMTS13 antigen were reduced, whereas VWF antigen was elevated among septic patients compared to non-septic patients and healthy controls. Non-septic patients also exhibited elevated VWF antigen and reduced ADAMTS13 activity, but to a lesser extent than septic patients. Non-survivor septic patients exhibited the lowest levels of ADAMTS13 activity. ADAMTS13 activity:antigen ratio was similar across all patient cohorts suggesting that the specific activity of ADAMTS13 remains unchanged. Therefore, reduced ADAMTS13 function in circulation is likely due to a reduction in circulating levels. We suggest that massive release of VWF in response to inflammation consumes limited circulating ADAMTS13, resulting in the imbalance observed between VWF and ADAMTS13 among septic and to a lesser extent in non-septic ICU patients. Changes to ADAMTS13 did not correlate with myeloperoxidase or protein C levels. Reduced ADAMTS13 activity and antigen, and elevated VWF antigen observed among all patient cohorts on admission remained unchanged in survivors at ICU discharge. Prolonged reduction in ADAMTS13 activity and antigen in septic patients coincides with elevated levels of VWF. The persistent abnormalities in ADAMTS13 and VWF in sepsis patients discharged from the ICU may contribute to a sustained prothrombotic state.