Vitamin K-dependent Coagulation Proteins Research Articles

Goal-Directed Use of Prothrombin Complex Concentrates in Liver Transplantation: Is a Plasma-Free Procedure Feasible?

Background: Fresh frozen plasma (FFP) transfusions have been the mainstay of hemostatic intervention for the treatment of bleeding and coagulation abnormalities arising during liver transplantation (LT) for decades. However, numerous clinical studies showed that FFP has many side effects, including the risk of pathogen transmission, transfusion-associated circulatory overload (TACO), transfusion-related immunomodulation (TRIM), and transfusion-related acute lung injury (TRALI). These adverse events are particularly challenging in patients undergoing LT, who often suffer from severe portal hypertension, poor renal function and coexisting cardiac disease.The aims of this review are to summarize the pharmacological properties of currently available PCCs, to represent the theoretical benefits and the possible risks related to the use of these drugs in patients undergoing LT, and, finally, to review the current literature on the topic in order to highlight the evidence that currently supports PCC use in LT patients. Methods: The current literature on the topic was reviewed in order to highlight the evidence that currently supports PCC use in LT patients. Results: Prothrombin complex concentrates (PCCs) may offer several advantages when compared to FFP. Indeed, PCCs have been shown to reduce the risk of TACO, which during liver transplantation may deteriorate portal hypertension, increase intraoperative bleeding, and possibly reduce survival rates. One of the major concerns for PCC use is thrombogenicity. However, currently available PCCs are much safer as they contain inactivated forms of the vitamin K-dependent coagulation factors and protein C, protein S, antithrombin and/or heparin. Nowadays, the use of PCCs to correct coagulation abnormalities that occur during LT is an increasingly widespread practice. However, it is not yet clear what level of evidence supports this practice, and what the risks associated with it are. Conclusions: Administration of PCC in LT patients to correct haemostatic abnormalities seems to be well-tolerated, but the relationship between PCC use and thromboembolic events in the postoperative period remains unclear. Adequately powered, methodologically sound trials are urgently required for more definitive conclusions about the efficacy and safety of PCCs in a broad phenotype of LT recipients.

The Plasma Proteomic Landscape of Thrombotic Events across Multi-Center Cohorts

Background: Diagnosis and risk prediction of thrombosis is challenging due to nonspecific symptoms, diverse risk factors, symptom overlap with other conditions and the variability in thrombosis presentations. Little is known on the impact of thrombosis on the plasma proteome across the spectrum of its clinical representations. Objective: The aim of this study was to explore the plasma proteomic landscape across multi-center thrombosis cohorts. The focus was on identifying protein signatures representative of thrombosis in general or among patients at risk of thrombosis, those with a history of thrombosis, those at acute phase and those with COVID-19 associated thrombosis. Method: Using state-of-the-art mass spectrometry-based proteomics, we analyzed 420 plasma proteome samples both cross-sectional and longitudinal from 211 patients, including those at risk of thrombosis, in the acute thrombotic phase (cerebral venous sinus thrombosis patients) and with COVID-19-associated thrombosis (Figure 1). Linear models and unsupervised co-expression clustering were used to explore common and distinct protein signatures among the cohorts. Results: Across the analyzed samples 475 proteins were quantified, with 86% showing a CV below 30%, and we found a strong correlation between protein levels quantified by MS and ELISA. Plasma proteomic alterations exhibited variation across cohorts, with the most pronounced changes observed in COVID-19 patients. While co-expression analysis unveiled distinct protein clusters associated with SARS-CoV-2 signaling pathways (CRP, SAA1) and Vitamin K-dependent (VKA) coagulation proteins (F9, F2, F10, PROS, PROC, PROZ, C4BPA), we did not identify a specific protein cluster related to thrombosis (Figure 2). Notably, VKA-therapy was associated with decreased abundance of VKA-dependent coagulation proteins, with F10 and PROZ showing significant alterations between patients who experienced a thrombosis compared to healthy controls. We observed an inverse correlation between the International Normalized Ratio and abundance of VKA-dependent coagulation proteins, with an increased correlation of F2 and F9 in patients who experienced a thrombotic event. Additionally, our analysis also highlighted potential variations in multi-center cohorts during sample handling, emphasizing the importance of standardized protocols in ensuring the reliability and comparability of results. Conclusion: Using a comprehensive MS-based proteomics approach, we identified clear plasma proteomic signatures associated with VKA-treatment and COVID-19 , but no distinct thrombosis signature across multi-center thrombosis cohorts.

Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV–FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin–antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

Vitamin K antagonist anticoagulant usage is associated with increased incidence and progression of osteoarthritis

ObjectivesVitamin K is hypothesised to play a role in osteoarthritis (OA) pathogenesis through effects on vitamin K-dependent bone and cartilage proteins, and therefore may represent a modifiable risk factor. A...

Vitamin K2 Needs an RDI Separate from Vitamin K1.

Vitamin K and its essential role in coagulation (vitamin K [Koagulation]) have been well established and accepted the world over. Many countries have a Recommended Daily Intake (RDI) for vitamin K based on early research, and its necessary role in the activation of vitamin K-dependent coagulation proteins is known. In the past few decades, the role of vitamin K-dependent proteins in processes beyond coagulation has been discovered. Various isoforms of vitamin K have been identified, and vitamin K2 specifically has been highlighted for its long half-life and extrahepatic activity, whereas the dietary form vitamin K1 has a shorter half-life. In this review, we highlight the specific activity of vitamin K2 based upon proposed frameworks necessary for a bioactive substance to be recommended for an RDI. Vitamin K2 meets all these criteria and should be considered for a specific dietary recommendation intake.

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1.

Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ-carboxylation might be of interest. The expression and functional activity of vitamin K-dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ-carboxylation that is mediated by the enzymes γ-carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX-producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT-PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assay and coagulation test. In addition, to quantify γ-carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX-producing HEK cells, resulting in a 3.2-fold higher expression of functional FIX, which displayed a 1.4-fold enhanced specific activity. Moreover, a 3.9-fold enhanced recovery of fully γ-carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ-carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.

Management of Severe Bleeding in Patients Treated With Oral Anticoagulants: Proceedings Monograph From the Emergency Medicine Cardiac Research and Education Group-International Multidisciplinary Severe Bleeding Consensus Panel October 20, 2018.

In this Emergency Medicine Cardiac Research and Education Group (EMCREG)-International Proceedings Monograph from the October 20, 2018, EMCREG-International Multidisciplinary Consensus Panel on Management of Severe Bleeding in Patients Treated With Oral Anticoagulants held in Orlando, FL, you will find a detailed discussion regarding the treatment of patients requiring anticoagulation and the reversal of anticoagulation for patients with severe bleeding. For emergency physicians, critical care physicians, hospitalists, cardiologists, internists, surgeons, and family physicians, the current approach and disease indications for treatment with anticoagulants such as coumadin, factor IIa, and factor Xa inhibitors are particularly relevant. When a patient treated with anticoagulants presents to the emergency department, intensive care unit, or operating room with severe, uncontrollable bleeding, achieving rapid, controlled hemostasis is critically important to save the patient's life. This EMCREG-International Proceedings Monograph contains multiple sections reflecting critical input from experts in Emergency Cardiovascular Care, Prehospital Emergency Medical Services, Emergency Medicine Operations, Hematology, Hospital Medicine, Neurocritical Care, Cardiovascular Critical Care, Cardiac Electrophysiology, Cardiology, Trauma and Acute Care Surgery, and Pharmacy. The first section provides a description of the current indications for the treatment of patients using oral anticoagulants including coumadin, the factor IIa (thrombin) inhibitor dabigatran, and factor Xa inhibitors such as apixaban and rivaroxaban. In the remaining sections, the treatment of patients presenting to the hospital with major bleeding becomes the focus. The replacement of blood components including red blood cells, platelets, and clotting factors is the critically important initial treatment for these individuals. Reversing the anticoagulated state is also necessary. For patients treated with coumadin, infusion of vitamin K helps to initiate the process of protein synthesis for the vitamin K-dependent coagulation proteins II, VII, IX, and X and the antithrombotic protein C and protein S. Repletion of clotting factors for the patient with 4-factor prothrombin complex concentrate, which includes factors II (prothrombin), VII, IX, and X and therapeutically effective concentrations of the regulatory proteins (protein C and S), provides real-time ability to slow bleeding. For patients treated with the thrombin inhibitor dabigatran, treatment using the highly specific, antibody-derived idarucizumab has been demonstrated to reverse the hypocoagulable state of the patient to allow blood clotting. In May 2018, andexanet alfa was approved by the US Food and Drug Administration to reverse the factor Xa anticoagulants apixaban and rivaroxaban in patients with major bleeding. Before the availability of this highly specific agent, therapy for patients treated with factor Xa inhibitors presenting with severe bleeding usually included replacement of lost blood components including red blood cells, platelets, and clotting factors and 4-factor prothrombin complex concentrate, or if not available, fresh frozen plasma. The evaluation and treatment of the patient with severe bleeding as a complication of oral anticoagulant therapy are discussed from the viewpoint of the emergency physician, neurocritical and cardiovascular critical care intensivist, hematologist, trauma and acute care surgeon, hospitalist, cardiologist, electrophysiologist, and pharmacist in an approach we hope that the reader will find extremely practical and clinically useful. The clinician learner will also find the discussion of the resumption of oral anticoagulation for the patient with severe bleeding after effective treatment important because returning the patient to an anticoagulated state as soon as feasible and safe prevents thrombotic complications. Finally, an EMCREG-International Severe Bleeding Consensus Panel algorithm for the approach to management of patients with life-threatening oral anticoagulant-associated bleeding is provided for the clinician and can be expanded in size for use in a treatment area such as the emergency department or critical care unit.

Coagulation Factor Plasma Levels Following Administration of a 4-Factor Prothrombin Complex Concentrate for Rapid Vitamin K Antagonist Reversal in Japanese Patients

BackgroundFour-factor prothrombin complex concentrates (4F-PCCs) have been approved for urgent vitamin K antagonist reversal in Western countries for many years. Ethnicity and genetic variations between populations may influence the pharmacokinetic profile of 4F-PCC treatments.ObjectiveTo report plasma levels of vitamin K-dependent coagulation factors and proteins C and S in Japanese patients following administration of a 4F-PCC approved recently in Japan.MethodsThis was a subanalysis of a prospective, open-label, Phase IIIb study in Japanese patients requiring rapid vitamin K antagonist reversal owing to major bleeding (n = 6) or need for urgent surgery (n = 5). International normalized ratio and plasma levels of factors II, VII, IX, and X, and proteins C and S were measured before PCC infusion and at specific time points for the next 24 hours. Adverse events and serious adverse events were recorded up to Day 14 and 45, respectively.ResultsRapid increases in plasma concentrations 30 minutes following 4F-PCC infusion were seen for all factors and proteins C and S, with median concentrations compared with baseline increasing by ≥100% and 70% in the bleeding and surgical groups, respectively. A concurrent decrease in international normalized ratio was observed. Plasma levels for each factor and protein remained within physiologic levels throughout the assessment period. No relationship between thromboembolic events and elevated plasma levels was identified.ConclusionsAdministration of 4F-PCC in Japanese patients receiving vitamin K antagonist anticoagulation therapy resulted in rapid and sustained increases in plasma levels and was well tolerated, indicating that this treatment is effective for the urgent reversal of vitamin K antagonist therapy in this population.

Successful Pregnancy Outcome in a Patient with Protein S Deficiency: A Case Report

Protein S is a vitamin K-dependent anticoagulant and has a central role in the regulation of coagulation. The mechanism of action of protein S has been one of the least understood amongst the vitamin K-dependent coagulation proteins. A deficiency of protein S predisposes to recurrent thromboembolism and fetal loss. Here we report a case of protein S deficiency in a 28- year-old pregnant woman, who had a history of complete abortion at 22 of weeks of gestation and her 2nd pregnancy was managed properly with a successful fetal outcome.Birdem Med J 2016; 6(2): 134-135

VKORc1 Is Under-Expressed in Skeletal Muscle of Humans, Dogs and Mice: Potential Implications for Ectopic Coagulation Factor Expression in Pre-Clinical and Therapeutic Applications

Post-translational modifications of coagulation factors in the liver are essential for function. The vitamin K dependent coagulation proteins (VKCPs) require vitamin K to undergo gamma carboxylation of the glutamic residues in their Gla domain by gamma-glutamyl carboxylase [GGCX]. The vitamin K is then recycled by the action of epoxide reductase [VKORc1] and/or quinone reductase [NQO1]. The hemostatic importance of the vitamin K “cycle” is evidenced by patients who may suffer bleeding complications when anticoagulated with warfarin, which targets the vitamin K cycle. Moreover, the ability of a variety of VKCPs to secrete a biologically active product depends on the removal of their propeptide by the action of the intracellular endoprotease furin [FURIN gene]. Previous in vitro work on recombinant coagulation Factor IX, which is used for hemophilia B treatment, has connected these two processing steps by showing that endogenous VKORc1 as well as FURIN can be limiting factors in high-yield expression systems. In vivo, skeletal muscle (in contrast to liver) has been utilized to express low levels of coagulation Factor IX in the first hemophilia B gene therapy clinical trial. However, our experiments in mice demonstrated that the specific activity of muscle-synthesized Factor IX via gene transfer decreased at the high levels of FIX expression by a limited muscle area (Schuettrumpf J. et al., Blood 2005). These results suggest that in vitro and in vivo expression of biologically-active VKCPs outside the liver may be limited by the host cell post-translational modification machinery. Here, we performed a systematic study to determine the expression profiles of the vitamin K cycle and furin endoprotease genes in human liver and muscle, compared to the mouse. We also established these profiles in two hemophilic dogs, given the extensive use of this animal model in gene-based hemophilia therapies. RNA from liver and skeletal muscle was used as a template for reverse transcription and the subsequent relative quantification of the GGCX, VKORc1, NQO1, and FURIN genes by qPCR in each tissue using a housekeeping reporter gene. For this, a variety of housekeeping genes were investigated in all three species to identify ones with similar transcript levels in both liver and muscle tissue. We identified the housekeeping genes HPRT1, beta actin, and 18s rRNA as equivalently expressed in the liver and skeletal muscle of human, mouse, and dog, respectively. The relative mRNA transcript quantification of the vitamin K cycle genes in humans showed that the transcript levels of GGCX were similar in liver and muscle. In contrast, both VKORc1 and NQO1 were under-expressed in muscle vs. liver (69.5 ± 4.9% and 67.8 ± 12.5%, respectively, P<0.01). In the mouse, VKORc1 transcript levels in the muscle were reduced to 73.8 ± 9.9% vs. liver (P<0.05), while GGCX and NQO1 exhibited similar transcript levels in both tissues. In the dog, we observed a dramatic reduction in VKORc1 and GGCX transcript levels in the muscle vs. liver (11.8 ± 4.2% and 29.5 ± 15.8%, respectively, P<0.01). Surprisingly, NQO1 transcript levels were 253.8 ± 156.7% higher in muscle than liver (P<0.05). Lastly, in all three species tested, transcript levels for FURIN were similar in both muscle and liver. Our results indicate that VKORc1, a key enzyme in the vitamin K cycle, is consistently under-expressed in the skeletal muscle of humans as well as in mice and hemophilic dogs. In contrast, FURIN transcripts are similarly abundant in the liver and muscle of all three species tested. These suggest that the vitamin K cycle but not propeptide processing by furin can be a limiting factor in the secretion of biologically active muscle-expressed VKCPs. As a result, our observations provide (1) a plausible explanation for the inverse relationship between specific activity and Factor IX expression levels in mice following Factor IX gene transfer, and (2) further support for the mouse and dog as useful models for therapies that depend on the muscle-derived expression of VKCPs. DisclosuresNo relevant conflicts of interest to declare.

The new oral anticoagulants: results of clinical trials and practical applications

The coumarin-type oral anticoagulants or vitamin K antagonists (VKA) have been in use since the early 1940s. Their discovery evolved from investigations into a hemorrhagic disease of cattle in the early twentieth century attributed to the consumption of spoiled sweet clover [1]. Link, a biochemist at the University of Wisconsin, isolated the responsible agent in spoiled sweet clover, dicoumarol(3-30 methyl-bis-4-hydroxy coumarin), which quickly entered the clinical arena in 1941. Link subsequently synthesized a related compound (warfarin), initially popularized as a rodenticide in the late 1940s, that entered clinical practice in the 1950s and quickly became the major oral anticoagulant in clinical use. Little has changed in the formulation of the coumarin-type oral anticoagulants. They have remained critically important drugs in the primary and secondary prevention of thromboembolism. Despite their widespread use, the vitamin K antagonists suffer from a number of problems that lead to underuse and inadequate treatment. Warfarin is safe and effective if a therapeutic international normalized ratio (INR) is properly maintained. In practice, however, it is difficult to manage because its therapeutic level is affected by many factors including diet, medications, illnesses, and genetics [2]. Warfarin’s narrow therapeutic range predisposes to many adverse events from both under anticoagulation (resulting in thrombus formation) and over anticoagulation (leading to hemorrhage). To ensure that a therapeutic INR is achieved, frequent monitoring is required that may be inconvenient for patients and physicians and costly for the healthcare system. Additional warfarin drawbacks include its slow onset and offset of action. It takes 72–96 h to become effective and requires overlap with a rapidly acting parenteral anticoagulant or ‘‘bridging’’ until a therapeutic INR is achieved. Its slow offset, with an effective half-life of approximately 40 h, makes it difficult to manage before procedures. Safety concerns and therapeutic complexity lead many physicians to underuse warfarin, prescribing it to only two-thirds of appropriate candidates [3]. Over the last decade a number of new oral anticoagulants have been developed and are now emerging from Phase III clinical trials [4]. Many of these agents are targeted to interact directly with, and inhibit a specific coagulation factor. This is in contrast to the VKAs that work indirectly by interfering with the synthesis of functional vitamin K-dependent coagulation proteins, thus, reducing their plasma concentration. Although commonly referred to as new or novel oral anticoagulants (NOACs), these drugs will quickly lose their newness and novelty. A more appropriate designation that denotes their mechanism of action (vis a vis VKAs) is ‘‘target-specific oral anticoagulants’’ or TSOAs. This designation will be used interchangeably with NOACs throughout this special issue. The target-specific oral anticoagulants in development are free from many of warfarin’s drawbacks and offer a convenient alternative to warfarin. Direct thrombin and direct Xa inhibitors are currently the most promising agents. By inhibiting these factors in the final common pathway of coagulation, these agents block both the intrinsic and extrinsic pathways. These small molecules directly block either factor Xa or IIa (thrombin) at their catalytic pocket. They promise to be more convenient and possibly safer than warfarin because they are given in fixed doses, have a predictable anticoagulant effect, do not require monitoring, have few or minimal interactions with drugs or diet, and have a rapid onset of action that eliminates the need for J. Ansell (&) New York University School of Medicine, New York, NY, USA e-mail: ansellje@gmail.com

VKORC1 C1173T and VKORC1 G-1639A Gene Polymorphisms in Turkish Behçet’s Patients with Ocular and Non-ocular Involvement

Background: Behçet’s disease (BD) is a multisystemic vasculitis with unknown etiology. Vitamin K epoxide reductase complex subunit 1 (VKORC1) is the key enyzme in the formation of active vitamin K that is a cofactor of various coagulation factors. Polymorphisms of the VKORC1 may affect the levels of active forms of vitamin K-dependent coagulation proteins and the tendency to thrombosis. The current study aimed to evaluate the role of VKORC1 gene polymorphisims in ocular and non-ocular Behçet’s disease.Methods: VKORC1 C1173T (rs 9934438) and G-1639A (rs 9923231) gene polymorphisms were evaluated by real-time polymerase chain reaction-based DNA analysis. The frequency of alleles and distribution of genotypes were assessed by the chi-squared test. Genotype distribution and Hardy–Weinberg equilibrium were tested with the χ2 test for quality of fit.Results: The distribution of GG, GA and AA and CC, CT, TT genotypes and the frequency of G,A and C,T alleles were not found to be different between patients and controls (p = 0.5651; p = 0.335 respectively), as well as patients with or without eye involvement (p = 0.9267; p = 0.384 respectively).Conclusion: VKORC1 polymorphisms seem not to be related with the thrombotic state of systemic and ocular Behçet’s disease.

Approach to the treatment, characterization and diagnosis of an acquired auto‐antibody directed against factors prothrombin, factor X and factor IX: a case report and review of the literature

Bleeding disorders secondary to acquired non-inhibitory antibodies directed against vitamin K-dependent coagulation proteins are rare. In this report, the authors describe a patient with a low grade lymphoma who presented with a fatal acquired bleeding manifestation and abnormal hemostatic studies resulting from deficiencies in both prothrombin and factor X. Patient plasma samples were collected and studied for the presence of an acquired inhibitor. Levels of plasma coagulation proteins were measured using immunoassay. Patient anti-prothrombin immunoglobulin G was isolated and binding to prothrombin, prothrombin F1.2, factors IX and X was evaluated using immunoblots and competition immunoassay. Prolongation in the prothrombin time and activated partial thromboplastin time suggested a factor deficiency in the common pathway of coagulation. Functional and antigenic levels of both prothrombin and factor X were decreased. An IgG subtype-4 antibody was isolated from patient plasma using affinity chromatography on prothrombin-sepharose. This antibody was found to bind to a common metal-ion-dependent conformational epitope found on the γ-carboxyglutamic acid (Gla) domain of prothrombin, factor X and factor IX. This report represents the first description of an acquired bleeding disorder resulting from a unique cross-reactive auto-antibody against a common metal-ion-dependent antigenic structure on the Gla-domain of the vitamin K-dependent proteins.

Ex Vivo Thrombin Generation in Patients With Venous Thromboembolic Disease or Atrial Fibrillation on Long-Term Oral Anticoagulation

International normalized ratio (INR) is used to monitor chronic oral anticoagulant (OA) treatment; however, it is poorly understood how this simple test reflects in vivo hemostatic reactions, culminating in thrombin generation and clot formation. We studied the process of thrombin generation using an ex vivo model, where thrombin-antithrombin (TAT) complexes are measured in blood emerging from standardized skin incisions in 55 patients (35 with venous thromboembolism [VTE] and 20 with sustained atrial fibrillation [AF]) treated with acenocoumarol (INR 2.0-3.0). In addition, in venous blood, we measured the activity of factor VIII (FVIII) and vitamin K-dependent coagulation proteins. Chronic anticoagulation led to significant reductions in maximum TAT concentrations as compared to 35 healthy controls, in maximum TAT generation rates, and in mean amount of thrombin generated. Parameters of thrombin generation did not correlate with INR or any coagulation factor measured. International normalized ratio was significantly and independently affected by the decrease in the activity of all vitamin K-dependent coagulation proteins. The strongest influence was shown for FVII. Factor VIII activity was increased in all patients studied independently of the duration of anticoagulation and did not change over time. In conclusion, in patients with VTE and AF on OA, there is no correlation between INR values and parameters of ex vivo thrombin generation. This may indicate an important role of protein C (PC) system and possibly other endothelium-dependent mechanisms in controlling hemostasis. Increased FVIII activity in patients with VTE and AF does not change significantly during anticoagulation and is probably related to the pretreatment prothrombotic state.

Zinc Binding to Protein C and Activated Protein C Modulates Their Interaction with Endothelial Cell Protein C Receptor.

Abstract 331▪▪This icon denotes an abstract that is clinically relevant. Introduction/background:Zinc is a multi-functional element that is essential for life and the second most abundant metal ion, after iron in eukaryotic organisms. Zinc deficiency has been associated with bleeding disorders and with defective platelet aggregation, suggesting it may play an important role in hemostasis. Zinc ions have been shown to enhance activation of the intrinsic pathway of coagulation but to down-regulate the extrinsic pathway of coagulation. All vitamin K-dependent coagulation proteins have calcium binding sites and may therefore to some extent, interact with other divalent metal ions, including zinc, through these sites. Recent crystallography studies identified a pair of Zn2+ binding sites in FVIIa protease domain, and with the exception of Glu220, all the side chains involved in both the Zn1 and Zn2 coordination in FVIIa are unique to FVIIa and are not present in other vitamin K-dependent clotting factors (Bajaj et al., J Biol Chem 2006; 281:24873-88). Nonetheless, Zn2+ may bind to other vitamin K-dependent clotting factors at sites different from those identified in FVIIa. Objective:The aim of the present study is to investigate the effect of zinc ions on the protein C pathway, particularly on protein C/APC binding to EPCR, protein C activation and APC catalytic activity. Methods:Protein C and APC binding to EPCR on endothelial cells was examined by radioligand binding studies. Protein C activation and APC catalytic activity were evaluated in chromogenic assays. Equilibrium dialysis was used to measure zinc binding to protein C/APC. Conformational changes in protein C/APC were monitored by intrinsic fluorescence quenching. Results:Zn2+ does not replace the Ca2+ as a mandatory cofactor for protein C/APC binding to EPCR but Zn2+ at physiologically relevant concentrations (10 to 25 μM) markedly increased Ca2+-dependent protein C and APC binding to EPCR (∼2 to 5-fold). The kinetic analysis of protein C and APC binding to EPCR suggested that Zn2+ enhanced protein C/APC binding to EPCR by increasing the binding affinity of protein C/APC to its receptor (Kd for APC: – Zn2+, 117 ± 27 nM; + Zn2+, 9.3 ± 3.3 nM; Kd, for protein C: – Zn2+, 96 ± 26 nM; + Zn2+, 21.4 ± 6.6 nM). The enhancing effect of Zn2+ on APC binding to EPCR was also observed in the presence of physiological concentrations of Mg2+, which itself increased the APC binding to EPCR, two-fold. Zn2+-mediated increased protein C binding to EPCR resulted in increased APC generation. The effect of Zn2+ was not limited to enhancing protein C and APC binding to EPCR but also affected the catalytic activity of APC. Zn2+ inhibited the amidolytic activity of APC half-maximally at 50 to 100 μM. Zn2+ also inhibited the amidolytic activity of Gla domain deleted (GD)-APC in a similar fashion. The inhibitory effect of Zn2+ was partially reversed by physiological concentrations of calcium. Addition of Zn2+ to protein C or APC quenched the intrinsic fluorescence of both APC and GD-APC. Data from the equilibrium binding studies performed with 65Zn2+ revealed that Zn2+ binds to both GD-APC and APC, but that the amount of Zn2+ bound to APC was 3 to 4-fold higher than the amount bound to GD-APC. Kinetic analysis of equilibrium binding studies suggested that two Zn2+ atoms bind to APC outside the Gla domain with relatively high affinity (∼70 μM). At least one of the Zn2+ sites may overlap with the Ca2+ binding site as the Zn2+ binding to GD-APC was inhibited by approximately 50% by saturating concentrations of Ca2+. The substantially increased Zn2+ binding to the APC compared to GD-APC suggested that the N-terminus of the Gla domain of protein C contains multiple Zn2+ binding sites. Interestingly, Zn2+ bound to APC and GD-APC with a similar high affinity suggesting that the Gla domain, as well as the protease domain, may contain high affinity binding sites for Zn2+. A majority of the Zn2+ binding sites in the Gla domain appear to be distinct from the Ca2+ binding sites as less than 40% of the maximal Zn2+ binding could be blocked by Ca2+. The putative zinc binding sites in protein C/APC appeared to be unique as no consensus canonical zinc binding sequences homologous to other known zinc binding proteins were found in protein C. Conclusions:Our present data show that Zn2+ binds to protein C/APC and induces a conformational change in these proteins, which in turn leads to higher affinity binding to their cellular receptor EPCR. Overall our results suggest that zinc ions may play an important regulatory role in the protein C pathway. Disclosures:No relevant conflicts of interest to declare.

Pleiotropic role of growth arrest-specific gene 6 in atherosclerosis

Growth arrest-specific gene 6 (Gas6) belongs to the family of vitamin K-dependent coagulation proteins, but in contrast to its other members, has only a limited role in hemostasis. Instead, Gas6 plays a prominent role in conditions of injury, inflammation and repair. Gas6 amplifies the activation of various cell types including endothelial cells and platelets in different models of thrombosis and inflammation, processes also important in atherosclerosis. Recently, we showed that in human and murine atherosclerotic plaques, Gas6 is expressed by endothelial cells, smooth muscle cells and most abundantly by macrophages, and that its expression increases with atherosclerosis severity. Moreover, genetic loss of Gas6 in ApoE mice reduced the influx of inflammatory cells in the plaque and induced plaque fibrosis, hence creating a stable plaque phenotype. Consistent herewith, Gas6 plasma levels are increased in patients with unstable angina pectoris, which is a common consequence of atherosclerotic plaque rupture. Inhibition of Gas6 would be an attractive therapeutic target for stabilizing atherosclerotic plaques and for the prevention of vascular thrombotic occlusion after plaque rupture. Here we will critically review the existing literature on the potential roles of Gas6 and its receptors in the different stages of atherosclerosis.

Regulation of coagulation by protein S

Protein S has been one of the least mechanistically understood amongst the vitamin K-dependent coagulation proteins, and diagnosis of protein S deficiency and quantification of the associated thrombotic risk are not straightforward. In this review, the regulation of thrombin generation by protein S and the pathophysiological implications of protein S deficiency are discussed in the light of recent findings on the anticoagulant function(s) of protein S. Protein S expresses both activated protein C-dependent and activated protein C-independent anticoagulant activities, but the former is generally believed to be lost upon binding of protein S to C4b-binding protein. Recently it has been shown that protein S acts as a cofactor of tissue factor pathway inhibitor in the down regulation of factor X-activation, which provides a mechanistic basis for the activated protein C-independent anticoagulant activity of protein S in plasma. In addition, reevaluation of the role of the protein S/C4b-binding protein complex has demonstrated that C4b-binding protein-bound protein S does express activated protein C-cofactor activity, especially during the inactivation of factor Va Leiden. These findings underscore the central role of protein S in the regulation of coagulation and may have important implications for the evaluation of the thrombotic risk associated with protein S deficiency.

Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif

Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-l-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7A resolution. The bovine Lact-C2 structure has a beta-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the beta-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-l-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.

Protein Z and natural anticoagulants in children on peritoneal dialysis and hemodialysis

Hemostatic alterations due to abnormalities in the coagulation and fibrinolytic system may occur in dialysis patients. Protein Z (PZ) is a vitamin K-dependent coagulation protein promoting assembly of thrombin with phospholipid vesicles. The aim of this study was to investigate PZ and natural anticoagulants in children on hemodialysis (HD) and peritoneal dialysis (PD). Protein Z, protein C (PC), protein S (PS), antithrombin III (AT III), and fibrinogen levels were studied in 24 PD, 13 HD patients and 23 controls. Plasma PZ levels in patients on HD were significantly higher than those on PD and control group (p = 0.04, p = 0.03). We observed elevated PC, PS and AT III activities in children on PD when compared to controls (p = 0.011, p = 0.003, p < 0.001). In HD patients, only PS activity was increased compared to controls (p = 0.016). PC and PS activities did not differ between PD and HD patients whereas AT III activity was higher in PD patients compared to HD patients (p < 0.001). Normal/high levels of PC, PS and AT III suggest that children on PD or HD treatment do not seem to have an increased risk of thrombogenesis due to reduction of these proteins. Increased PZ levels, however, might contribute to the hemostatic alterations in children on HD treatment along with other well known abnormalities.

Functional promoter polymorphism in the VKORC1 gene is no major genetic determinant for coronary heart disease in Northern Germans

Recently, the C-allele of polymorphism rs2359612 (VKORC1: c.283+837C>T) in the VKORC1 gene has been reported to represent a major risk factor for coronary heart disease (CHD), stroke, and aortic dissection in Chinese patients. VKOR activity itself is the rate-limiting step in gamma-carboxylation of vitamin K-dependent coagulation factors (factors II, VII, IX, X, protein C, S, and Z) and proteins of calcium metabolism (matrix Gla protein and osteocalcin). Gamma-carboxylation is essential for the biological activity of these proteins that have been previously hypothesised to play a role in the pathogenesis of atherosclerosis. It was the objective of this study to analyse the VKORC1 genotype frequency in patients with CHD and controls from Northern Germany and to investigate the association of VKORC1 and CHD risk in patients with an European background. CHD patients (n = 901) and healthy controls (n = 521) were part of the PopGen biobank. Case and control samples were matched for ethnic and geographic origin, age and gender. After typing German CHD patients and control individuals, no evidence for a statistically significant association was detected between VKORC1 genotype and CHD phenotype. Also stratification for gender and myocardial infarction yielded no significant results. In conclusion, the discrepant association findings in Chinese and German populations may be explained by ethnic differences in genetic and perhaps environmental predisposition, modifying the polygenic CHD phenotype by interacting with VKORC1 variants and thus conferring disease susceptibility in some populations, but not in others.