Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration.

ABSTRACT: Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Helix unwinding and base flipping enable human MTERF1 to terminate mitochondrial transcription.

Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that the mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.

Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression.

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.

Mapping proteolytic neo-N termini at the surface of living cells.

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2.

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses.

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85ß subunit. Here we present the mechanism underlying the molecular recognition of the p85ß subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85ß of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85ß. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1ED exists in a dynamic equilibrium between p85ß-binding-competent and -incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85ß. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1ED can result in the faster hijacking of p85ß compared to Ud NS1ED, although the former has a lower affinity to p85ß than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

DNA origami protection and molecular interfacing through engineered sequence-defined peptoids.

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson-Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1-9) with two types of architectures, termed as "brush" and "block," were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

A high-fat diet delays plasmin generation in a thrombomodulin-dependent manner in mice.

Obesity is a prevalent prothrombotic risk factor marked by enhanced fibrin formation and suppressed fibrinolysis. Fibrin both promotes thrombotic events and drives obesity pathophysiology, but a lack of essential analytical tools has left fibrinolytic mechanisms affected by obesity poorly defined. Using a plasmin-specific fluorogenic substrate, we developed a plasmin generation (PG) assay for mouse plasma that is sensitive to tissue plasminogen activator, α2-antiplasmin, active plasminogen activator inhibitor (PAI-1), and fibrin formation, but not fibrin crosslinking. Compared with plasmas from mice fed a control diet, plasmas from mice fed a high-fat diet (HFD) showed delayed PG and reduced PG velocity. Concurrent to impaired PG, HFD also enhanced thrombin generation (TG). The collective impact of abnormal TG and PG in HFD-fed mice produced normal fibrin formation kinetics but delayed fibrinolysis. Functional and proteomic analyses determined that delayed PG in HFD-fed mice was not due to altered levels of plasminogen, α2-antiplasmin, or fibrinogen. Changes in PG were also not explained by elevated PAI-1 because active PAI-1 concentrations required to inhibit the PG assay were 100-fold higher than circulating concentrations in mice. HFD-fed mice had increased circulating thrombomodulin, and inhibiting thrombomodulin or thrombin-activatable fibrinolysis inhibitor (TAFI) normalized PG, revealing a thrombomodulin- and TAFI-dependent antifibrinolytic mechanism. Integrating kinetic parameters to calculate the metric of TG/PG ratio revealed a quantifiable net shift toward a prothrombotic phenotype in HFD-fed mice. Integrating TG and PG measurements may define a prothrombotic risk factor in diet-induced obesity.

Tools for supporting solution scattering during the COVID-19 pandemic.

During the COVID-19 pandemic, synchrotron beamlines were forced to limit user access. Performing routine measurements became a challenge. At the Life Science X-ray Scattering (LiX) beamline, new instrumentation and mail-in protocols have been developed to remove the access barrier to solution scattering measurements. Our efforts took advantage of existing instrumentation and coincided with the larger effort at NSLS-II to support remote measurements. Given the limited staff-user interaction for mail-in measurements, additional software tools have been developed to ensure data quality, to automate the adjustments in data processing, as users would otherwise rely on the experience of the beamline staff, and produce a summary of the initial assessments of the data. This report describes the details of these developments.

Coagulation factor XIIIa cross-links amyloid ß into dimers and oligomers and to blood proteins.

In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid ß (Aß) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aß in CAA, the ability of FXIIIa to cross-link Aß has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aß40 into dimers and oligomers (kcat/Km = 1.5 × 105 m-1s-1), as well as to fibrin, platelet proteins, and blood clots under flow in vitro Aß40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that FXIIIa-mediated cross-linking may contribute to the formation of Aß deposits in CAA and Alzheimer's disease.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

Solution scattering at the Life Science X-ray Scattering (LiX) beamline.

This work reports the instrumentation and software implementation at the Life Science X-ray Scattering (LiX) beamline at NSLS-II in support of biomolecular solution scattering. For automated static measurements, samples are stored in PCR tubes and grouped in 18-position sample holders. Unattended operations are enabled using a six-axis robot that exchanges sample holders between a storage box and a sample handler, transporting samples from the PCR tubes to the X-ray beam for scattering measurements. The storage box has a capacity of 20 sample holders. At full capacity, the measurements on all samples last for ∼9 h. For in-line size-exclusion chromatography, the beamline-control software coordinates with a commercial high-performance liquid chromatography (HPLC) system to measure multiple samples in batch mode. The beamline can switch between static and HPLC measurements instantaneously. In all measurements, the scattering data span a wide q-range of typically 0.006-3.2 Å-1. Functionalities in the Python package py4xs have been developed to support automated data processing, including azimuthal averaging, merging data from multiple detectors, buffer scattering subtraction, data storage in HDF5 format and exporting the final data in a three-column text format that is acceptable by most data analysis tools. These functionalities have been integrated into graphical user interfaces that run in Jupyter notebooks, with hooks for external data analysis software.

Red blood cells in thrombosis.

Red blood cells (RBCs) have historically been considered passive bystanders in thrombosis. However, clinical and epidemiological studies have associated quantitative and qualitative abnormalities in RBCs, including altered hematocrit, sickle cell disease, thalassemia, hemolytic anemias, and malaria, with both arterial and venous thrombosis. A growing body of mechanistic studies suggests that RBCs can promote thrombus formation and enhance thrombus stability. These findings suggest that RBCs may contribute to thrombosis pathophysiology and reveal potential strategies for therapeutically targeting RBCs to reduce thrombosis.

A Low-Fidelity, High-Functionality Ultrasound-Guided Serratus Plane Block Model.

BACKGROUND: Thoracic injuries present many challenges for management in the acute and inpatient settings, including achieving appropriate pain control. Traditional modalities, such as opioids and spinal epidural anesthesia, are associated with multiple complications. Ultrasound-guided regional nerve blocks are becoming more prevalent, and they have been shown to be an effective modality of pain control for other traumatic injuries. Models comprised of animal tissue to simulate human anatomy are widely utilized to facilitate training of needle-guided procedures, but no such model for the serratus anterior plane block has yet been defined in the literature. OBJECTIVES: Our goal was to produce a high-functionality serratus anterior plane block model with reasonable anatomic fidelity from low-cost materials. DISCUSSION: We describe the creation of an inexpensive high-functionality serratus anterior plane block model from common materials, including pork ribs and chicken breasts, to realistically simulate human anatomy, including multiple muscle and fascial planes, as well as to allow hydrodissection. CONCLUSIONS: This model will facilitate training and can improve success when caring for patients with thoracic trauma.

The interaction between fibrinogen and zymogen FXIII-A2B2 is mediated by fibrinogen residues γ390-396 and the FXIII-B subunits.

Coagulation transglutaminase factor XIII (FXIII) exists in circulation as heterotetrameric proenzyme FXIII-A2B2 Effectively all FXIII-A2B2 circulates bound to fibrinogen, and excess FXIII-B2 circulates in plasma. The motifs that mediate interaction of FXIII-A2B2 with fibrinogen have been elusive. We recently detected reduced binding of FXIII-A2B2 to murine fibrinogen that has γ-chain residues 390-396 mutated to alanines (Fibγ390-396A). Here, we evaluated binding features using human components, including recombinant fibrinogen variants, FXIII-A2B2, and isolated FXIII-A2 and -B2 homodimers. FXIII-A2B2 coprecipitated with wild-type (γA/γA), alternatively-spliced (γ'/γ'), and αC-truncated (Aα251) fibrinogens, whereas coprecipitation with human Fibγ390-396A was reduced by 75% (P <0001). Surface plasmon resonance showed γA/γA, γ'/γ', and Aα251 fibrinogens bound FXIII-A2B2 with high affinity (nanomolar); however, Fibγ390-396A did not bind FXIII-A2B2 These data indicate fibrinogen residues γ390-396 comprise the major binding motif for FXIII-A2B2 Compared with γA/γA clots, FXIII-A2B2 activation peptide release was 2.7-fold slower in Fibγ390-396A clots (P < .02). Conversely, activation of recombinant FXIII-A2 (lacking FXIII-B2) was similar in γA/γA and Fibγ390-396A clots, suggesting fibrinogen residues γ390-396 accelerate FXIII-A2B2 activation in a FXIII-B2-dependent mechanism. Recombinant FXIII-B2 bound γA/γA, γ'/γ', and Aα251 with similar affinities as FXIII-A2B2, but did not bind or coprecipitate with Fibγ390-396A FXIII-B2 also coprecipitated with fibrinogen from FXIII-A-deficient mouse and human plasmas. Collectively, these data indicate that FXIII-A2B2 binds fibrinogen residues γ390-396 via the B subunits, and that excess plasma FXIII-B2 is not free, but rather circulates bound to fibrinogen. These findings provide insight into assembly of the fibrinogen/FXIII-A2B2 complex in both physiologic and therapeutic situations.

Coagulation factor XIIIa is inactivated by plasmin.

Coagulation factor XIIIa (FXIIIa) is a transglutaminase that covalently cross-links fibrin and other proteins to fibrin to stabilize blood clots and reduce blood loss. A clear mechanism to describe the physiological inactivation of FXIIIa has been elusive. Here, we show that plasmin can cleave FXIIIa in purified systems and in blood. Whereas zymogen FXIII was not readily cleaved by plasmin, FXIIIa was rapidly cleaved and inactivated by plasmin in solution (catalytic efficiency = 8.3 × 10(3) M(-1)s(-1)). The primary cleavage site identified by mass spectrometry was between K468 and Q469. Both plasma- and platelet-derived FXIIIa were susceptible to plasmin-mediated degradation. Inactivation of FXIIIa occurred during clot lysis and was enhanced both in plasma deficient in fibrinogen and in plasma treated with therapeutic levels of tissue plasminogen activator. These results indicate that FXIIIa activity can be modulated by fibrinolytic enzymes, and suggest that changes in fibrinolytic activity may influence cross-linking of blood proteins.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking.

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.

Newly-Recognized Roles of Factor XIII in Thrombosis.

Arterial and venous thromboses are major contributors to coagulation-associated morbidity and mortality. Greater understanding of mechanisms leading to thrombus formation and stability is expected to lead to improved treatment strategies. Factor XIII (FXIII) is a transglutaminase found in plasma and platelets. During thrombosis, activated FXIII cross-links fibrin and promotes thrombus stability. Recent studies have provided new information about FXIII activity during coagulation and its effects on clot composition and function. These findings reveal newly-recognized roles for FXIII in thrombosis. Herein, we review published literature on FXIII biology and effects on fibrin structure and stability, epidemiologic data associating FXIII with thrombosis, and evidence from animal models indicating FXIII has an essential role in determining thrombus stability, composition, and size.

Elevated prothrombin promotes venous, but not arterial, thrombosis in mice.

OBJECTIVE: Individuals with elevated prothrombin, including those with the prothrombin G20210A mutation, have increased risk of venous thrombosis. Although these individuals do not have increased circulating prothrombotic biomarkers, their plasma demonstrates increased tissue factor-dependent thrombin generation in vitro. The objectives of this study were to determine the pathological role of elevated prothrombin in venous and arterial thrombosis in vivo, and distinguish thrombogenic mechanisms in these vessels. APPROACH AND RESULTS: Prothrombin was infused into mice to raise circulating levels. Venous thrombosis was induced by electrolytic stimulus to the femoral vein or inferior vena cava ligation. Arterial thrombosis was induced by electrolytic stimulus or ferric chloride application to the carotid artery. Mice infused with prothrombin demonstrated increased tissue factor-triggered thrombin generation measured ex vivo, but did not have increased circulating prothrombotic biomarkers in the absence of vessel injury. After venous injury, elevated prothrombin increased thrombin generation and the fibrin accumulation rate and total amount of fibrin ≈ 3-fold, producing extended thrombi with increased mass. However, elevated prothrombin did not accelerate platelet accumulation, increase the fibrin accumulation rate, or shorten the vessel occlusion time after arterial injury. CONCLUSIONS: These findings reconcile previously discordant findings on thrombin generation in hyperprothrombinemic individuals measured ex vivo and in vitro, and show elevated prothrombin promotes venous, but not arterial, thrombosis in vivo.