The complex reactions of blood coagulation are balanced by several natural anticoagulants resulting in tuned hemostasis. During several decades, the knowledge base of the natural anticoagulants has ...greatly increased and we have also learned about antiinflammatory and cytoprotective activities expressed by antithrombin and activated protein C (APC). Some coagulation proteins have also been found to function as anticoagulants; e.g., thrombin when bound to thrombomodulin activates protein C. Another example is factor V (FV), which in addition to being a procofactor to FVa has emerged as an anticoagulant. The discovery of APC resistance, caused by FVLeiden, as a thrombosis risk factor resulted in the identification of FV as an APC cofactor working in synergy with protein S in the regulation of FVIIIa in the Xase complex. More recently, a natural anticoagulant FV splice isoform (FV-Short) was discovered when investigating the East Texas bleeding disorder. In FV-Short, the truncated B domain exposes a high-affinity binding site for tissue factor pathway inhibitor alpha (TFPIα), and together with protein S a high-affinity trimolecular complex is generated. The FXa-inhibitory activity of TFPIα is synergistically stimulated by FV-Short and protein S. The circulating FV-Short/protein S/TFPIα complex concentration is normally low (≈0.2 nM) but provides an anticoagulant threshold. In the East Texas bleeding, the concentration of the complex, and thus the threshold, is increased 10-fold, which results in bleeding manifestations. The anticoagulant properties of FV were discovered during investigations of individual patients and follow the great tradition of bed-to-bench and bench-to-bed research in the coagulation field.
Venous thromboembolism is a major medical problem, annually affecting 1 in 1000 individuals. It is a typical multifactorial disease, involving both genetic and circumstantial risk factors that affect ...a delicate balance between procoagulant and anticoagulant forces. In the last 50 years, the molecular basis of blood coagulation and the anticoagulant systems that control it have been elucidated. This has laid the foundation for discoveries of both common and rare genetic traits that tip the natural balance in favor of coagulation, with a resulting lifelong increased risk of venous thrombosis. Multiple mutations in the genes for anticoagulant proteins such as antithrombin, protein C, and protein S have been identified and constitute important risk factors. Two single mutations in the genes for coagulation factor V (FV Leiden) and prothrombin (20210G>A), resulting from approximately 20 000-year-old mutations with subsequent founder effects, are common in the general population and constitute major genetic risk factors for thrombosis. In celebration of the 50-year anniversary of the American Society of Hematology, this invited review highlights discoveries that have contributed to our present understanding of the systems that control blood coagulation and the genetic factors that are involved in the pathogenesis of venous thrombosis.
Background
Factor V‐Short (FV756‐1458) is a natural splice variant in which 702 residues are deleted from the B domain. It exposes an acid region (AR2; 1493–1537) that binds tissue factor pathway ...inhibitor alpha (TFPIα). Protein S also interacts with TFPIα and serves as TFPIα‐cofactor in factor Xa (FXa) inhibition. FV‐Short and protein S function as synergistic TFPIα‐cofactors in inhibition of FXa. FV810‐1492 is an artificial FV‐Short variant that cannot synergize with protein S as TFPIα cofactor even though it contains AR2 and binds TFPIα.
Objective
To elucidate the mechanisms for the synergism between FV756‐1458 and protein S as TFPIα cofactors.
Methods
Four FV‐Short variants were created, FV756‐1458 and FV712‐1458 contained the preAR2 region (1458–1492), whereas FV810‐1492 and FV713‐1492 lacked this region. The synergistic TFPIα cofactor activity between FV‐Short variants and protein S was analyzed by FXa‐inhibition. A microtiter‐based assay tested binding between FV‐Short variants, protein S, and TFPIα.
Results
The two preAR2‐containing FV‐Short variants were active as synergistic TFPIα cofactors, whereas the other two were inactive. All variants bound to TFPIα. None of the FV‐Short variants bound directly to protein S. The combination of TFPIα and preAR2‐containing FV‐Short variants bound protein S, whereas TFPIα together with the preAR2‐minus variants did not. Protein S potentiated TFPIα‐binding to the preAR2‐containing variants and binding between TFPIα and protein S was stimulated only by the preAR2‐containing variants.
Conclusion
The preAR2 region is demonstrated to be crucial for the synergistic TFPIα‐cofactor activity between FV‐Short and protein S and for the assembly of a trimolecular FXa‐inhibitory complex comprising FV‐Short, protein S, and TFPIα.
Background
Factor V‐short (FV756‐1458) is a natural splice variant functioning in synergy with protein S as tissue factor pathway inhibitor alpha (TFPIα)–cofactor in inhibition of factor Xa (FXa). An ...exposed acid region (AR2; 1493–1537) in the B domain binds TFPIα. The preAR2 (1458–1492) is crucial for the synergistic TFPIα–cofactor activity between FV‐short and protein S and for assembly of a trimolecular FXa‐inhibitory complex among FV‐short, protein S, and TFPIα.
Objective
To identify which part of preAR2 is required for the synergistic TFPIα–cofactor activity between FV‐short and protein S.
Methods
A FXa‐inhibition assay was used to test the synergistic TFPIα cofactor activity between protein S and new FV‐short variants FV709‐1476, FV712‐1478, FV712‐1481, FV712‐1484, FV712‐1487, and FV712‐1490. A microtiter‐based assay analyzed binding among FV‐short variants, protein S, and TFPIα.
Results
FV709‐1476, FV712‐1478, and FV712‐1481 were fully active as synergistic TFPIα cofactors with protein S; FV712‐1484 showed intermediate activity; and FV712‐1487 and FV712‐1490 were inactive. TFPIα interacted with all variants in the absence of protein S but FV712‐1478 and FV712‐1481 bound TFPIα with highest affinity. None of the FV‐short variants bound directly to protein S in the absence of TFPIα. In the presence of TFPIα, efficient cooperative binding was demonstrated between protein S, TFPIα, and FV709‐1476, FV712‐1478, or FV712‐1481. In contrast, no cooperativity among TFPIα, protein S, and FV712‐1484, FV712‐1487, or FV712‐1490 was seen.
Conclusion
A short hydrophobic patch in preAR2 (PLVIVG, 1481–1486) in FV‐short is crucial for the synergistic TFPIα‐cofactor activity between FV‐short and protein S and for the assembly of a trimolecular FXa‐inhibitory complex among FV‐short, protein S, and TFPIα.
Nanoparticles (NPs) are increasingly used in diagnostic and drug delivery. After entering the bloodstream, a protein corona will form around NPs. The size and curvature of NPs is one of the major ...characteristics affecting the composition of bound protein in the corona. Key initiators of the intrinsic pathway of blood coagulation, the contact activation complex, (Kallikrein, Factor XII, and high molecular weight Kininogen) have previously been identified on NPs surfaces. We show that the functional impact of carboxyl-modified polystyrene NPs on these initiators of the intrinsic pathway is size dependent. NPs with high curvature affect the enzymatic activity differently from NPs with low curvature. The size dependency is evident in full blood plasma as well as in solutions of single coagulation factors. NPs induce significant alteration of the enzymatic activity in a size-dependent manner, and enzyme kinetics studies show a critical role for NPs surface area and curvature.
In Cell Reports, Christoffersen et al. 1 demonstrate that sphingosine 1-phosphate (S1P) bound to apolipoprotein M (apoM) regulates the activity and mass of brown adipose tissue (BAT). They found mice ...lacking apoM to have hyperactive BAT with high triglyceride (TG) utilization, resulting in low white adipose tissue (WAT) mass and low body weight.
The protein corona formed around nanoparticles in protein-rich fluids plays an important role for nanoparticle biocompatibility, as found in several studies during the last decade. Biological fluids ...have complex compositions and the molecular components interact and function together in intricate networks. Therefore, the process to isolate blood or the preparation of blood derivatives may lead to differences in the composition of the identified protein corona around nanoparticles. Here, we show distinct differences in the protein corona formed in whole blood, whole blood with EDTA, plasma, or serum. Furthermore, the ratio between particle surface area to protein concentration influences the detected corona. We also show that the nanoparticle size per se influences the formed protein corona due to curvature effects. These results emphasize the need of investigating the formation and biological importance of the protein corona in the same environment as the nanoparticles are intended for or released into.
Summary
Factor V (FV) serves an important role in the regulation of blood coagulation, having both pro‐ and anticoagulant properties. The circulating high molecular weight single‐chain FV molecule ...undergoes a series of proteolytic cleavages during both activation of coagulation and during anticoagulant regulation of coagulation by activated protein C (APC). It is noteworthy that mutations in the factor V gene (F5) either cause thrombosis or bleeding. New insights into the importance and complexity of FV functions have been generated from elucidation of the pathogenic mechanisms of two familial mutations in the F5 gene. The first mutation was identified as a result of the discovery of APC resistance as the most common risk factor for venous thrombosis. The mutation (FV Leiden) predicts the Arg506Gln replacement, which impairs the normal regulation of FVa by APC, as the Arg506 site is an important APC cleavage site. In addition, elucidation of APC resistance resulted in the discovery of the anticoagulant APC cofactor activity of FV. The second FV mutation (FVA2440G), identified in a family with an autosomal dominant bleeding disorder, has led to the discovery of an alternative splicing generating a previously unidentified FV isoform (FV‐Short), which inhibits coagulation via an unexpected and intriguing mechanism involving the coagulation inhibitor TFPI‐α. These are naturally occurring mutations in the F5 gene that have generated new knowledge on the role of FV in regulation of coagulation and the importance of genetic risk factors for thrombosis and bleeding.
Overexpression of the receptor tyrosine kinase Axl is implicated in several cancers. Therefore, we conducted this study to determine the expression of Axl and its ligand Gas6 in various renal cell ...carcinoma (RCC) types and in oncocytoma.
Real-time quantitative reverse transcription-PCR was used to quantify tumor mRNA levels for Axl and Gas6 in a cohort (n = 221) of RCC patients. Serum levels of soluble sAxl and Gas6 proteins were measured using specific ELISA assays (n = 282). The presence of Axl protein in tumor tissue was evaluated by immunohistochemistry (n = 294). Results were correlated to tumor-associated variables, clinical biochemical tests, and patient survival.
Tumor Axl mRNA levels correlated independently to survival when assessed against tumor stage and grade. In the study group, the median cancer-specific survival of all RCC patients during 307 months of follow-up was 55 months (confidence interval, +/-40.4). The 25% of patients with lowest tumor Axl mRNA levels had significantly better survival than the rest (P = 0.0005), with 70% of the patients still alive at the end of follow-up. In contrast, in patients with medium-high Axl mRNA, only 25% were alive at the end of follow-up. Tumor Gas6 mRNA levels correlated to survival, tumor-associated variables, and disease severity as did serum levels of soluble sAxl and Gas6 protein. However, no correlation between Axl protein in tumor tissue and survival was found.
Axl and Gas6 expression in RCC are associated with tumor advancement and patient survival. In particular, low tumor Axl mRNA levels independently correlated with improved survival.
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