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.

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.

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.

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.

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.

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.

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.

Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape.

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure-function relationships for PAI-1 and for the interpretation of human genomic sequence variants.

Illustrated State-of-the-Art Capsules of the ISTH 2020 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress.

Diagnostic Potential of Coagulation-Related Biomarkers for Sepsis in the Emergency Department: Protocol for a Pilot Observational Cohort Study.

BACKGROUND: Between 75% and 80% of patients with sepsis arrive in the hospital through the emergency department. Early diagnosis is important to alter patient prognosis, but currently, there is no reliable biomarker. The innate immune response links inflammation and coagulation. Several coagulation -related biomarkers are associated with poor prognosis in the ICU. The role of coagulation biomarkers to aid in early sepsis diagnosis has not previously been investigated. The objective of our study is to determine the individual or combined accuracy of coagulation and inflammation biomarkers with standard biochemical tests to diagnose adult septic patients presenting to the emergency department. METHODS: in the Emergency Department is a prospective, observational cohort study with a target enrolment of 250 suspected septic patients from two Canadian emergency departments. The emergency physicians will enroll patients with suspected sepsis. Blood samples will be collected at two time points (initial presentation and 4 hr following). Patients will be adjudicated into septic, infected, or not infected status in accordance with the Sepsis-3 definitions. Patient demographics, cultures, diagnosis, and biomarkers will be reported using descriptive statistics. Optimal cut off values with sensitivity and specificity for each biomarker will be determined using C-statistics to distinguish between septic and nonseptic patients. Stepwise multiple logistic regression analysis with exclusion of nonsignificant covariates from the final model will be used to establish a panel of biomarkers. CONCLUSIONS: Our protocol describes the processes and methods for a pragmatic observational biomarker study in the emergency department. This study will seek to determine the potential diagnostic importance of early coagulation abnormalities to identify additional tools for sepsis diagnosis.

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.

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish.

The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >107 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

Mapping the Substrate Recognition Landscapes of Metalloproteases Using Comprehensive Mutagenesis.

Protease specificity is controlled by exosites, which capture and orient the substrate, and the active site, which binds substrate residues near the P1-P1' scissile bond and catalyzes peptide hydrolysis. Techniques used to identify critical contact points between a protease and its substrate can be time consuming and labor-intensive. Screening tools such as phage display have been revitalized with the emergence of high-throughput sequencing technology, and can be used to interrogate protease substrate specificity. This article will outline a method for creating and screening a comprehensive mutagenesis substrate phage display library. High-throughput sequencing of uncleaved phage at various reaction time points enables k cat/K M determination for every possible single amino acid substitution at each position of the substrate, providing unprecedented resolution for the interaction between a protease and its substrate.

Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in circulating blood, limiting the size of VWF multimers and regulating VWF activity. Abnormal regulation of VWF contributes to bleeding and to thrombotic disorders. ADAMTS13 levels in plasma are highly variable among healthy individuals, although the heritability and the genetic determinants of this variation are unclear. We performed genome-wide association studies of plasma ADAMTS13 concentrations in 3244 individuals from 2 independent cohorts of healthy individuals. The heritability of ADAMTS13 levels was between 59.1% (all individuals) and 83.5% (siblings only), whereas tobacco smoking was associated with a decrease in plasma ADAMTS13 levels. Meta-analysis identified common variants near the ADAMTS13 locus on chromosome 9q34.2 that were significantly associated with ADAMTS13 levels and collectively explained 20.0% of the variance. The top single nucleotide polymorphism (SNP), rs28673647, resides in an intron of ADAMTS13 (ß, 6.7%; P = 1.3E-52). Conditional analysis revealed 3 additional independent signals represented by rs3739893 (ß, -22.3%; P = 1.2E-30) and rs3124762 (ß, 3.5%; P = 8.9E-9) close to ADAMTS13 and rs4075970 (ß, 2.4%; P = 6.8E-9) on 21q22.3. Linkage analysis also identified the region around ADAMTS13 (9q34.2) as the top signal (LOD 3.5), consistent with our SNP association analyses. Two nonsynonymous ADAMTS13 variants in the top 2 independent linkage disequilibrium blocks (Q448E and A732V) were identified and characterized in vitro. This study uncovered specific common genetic polymorphisms that are key genetic determinants of the variation in plasma ADAMTS13 levels in healthy individuals.

Corrigendum to "Characterization of Zebrafish von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage".

[This corrects the article DOI: 10.1155/2012/214209.].

Modeling Disorders of Blood Coagulation in the Zebrafish.

Hemostasis, the process of blood clot formation and resolution in response to vascular injury, and thrombosis, the dysregulation of hemostasis leading to pathological clot formation, are widely studied. However, the genetic variability in hemostatic and thrombotic disorders is incompletely understood, suggesting that novel mediators have yet to be uncovered. The zebrafish is developing into a powerful in vivo model to study hemostasis, and its features as a model organism are well suited to (a) develop high-throughput screens to identify novel mediators of hemostasis and thrombosis, (b) validate candidate genes identified in human populations, and (c) characterize the structure/function relationship of gene products. In this review, we discuss conservation of the zebrafish hemostatic system, highlight areas for future study, and outline the utility of this model to study blood coagulation and its dysregulation.