A novel method to quantify fibrin-fibrin and fibrin-α2-antiplasmin cross-links in thrombi formed from human trauma patient plasma.

BACKGROUND: The widespread use of the antifibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, factor (F)XIIIa catalyzes the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links, which increases clot mechanical strength and resistance to fibrinolysis. OBJECTIVES: Here, we developed a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. METHODS: Fibrinogen alpha (FGA) chain (FGA-FGA), fibrinogen gamma (FGG) chain (FGG-FGG), and FGA-α2AP cross-links were quantified using liquid chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring in paired plasma samples from trauma patients prefibrinogen and postfibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analyzed. RESULTS: The abundance of cross-links was significantly increased in trauma patients postcryo, but not Fg-C transfusion (P < .0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibers, the peak thrombin concentration, and FXIII antigen (P < .05). CONCLUSION: We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach, we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared with Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients.

Cryoprecipitate transfusion in trauma patients attenuates hyperfibrinolysis and restores normal clot structure and stability: Results from a laboratory sub-study of the FEISTY trial.

BACKGROUND: Fibrinogen is the first coagulation protein to reach critical levels during traumatic haemorrhage. This laboratory study compares paired plasma samples pre- and post-fibrinogen replacement from the Fibrinogen Early In Severe Trauma studY (FEISTY; NCT02745041). FEISTY is the first randomised controlled trial to compare the time to administration of cryoprecipitate (cryo) and fibrinogen concentrate (Fg-C; Riastap) in trauma patients. This study will determine differences in clot strength and fibrinolytic stability within individuals and between treatment arms. METHODS: Clot lysis, plasmin generation, atomic force microscopy and confocal microscopy were utilised to investigate clot strength and structure in FEISTY patient plasma. RESULTS: Fibrinogen concentration was significantly increased post-transfusion in both groups. The rate of plasmin generation was reduced 1.5-fold post-transfusion of cryo but remained unchanged with Fg-C transfusion. Plasminogen activator inhibitor 1 activity and antigen levels and Factor XIII antigen were increased post-treatment with cryo, but not Fg-C. Confocal microscopy analysis of fibrin clots revealed that cryo transfusion restored fibrin structure similar to those observed in control clots. In contrast, clots remained porous with stunted fibres after infusion with Fg-C. Cryo but not Fg-C treatment increased individual fibre toughness and stiffness. CONCLUSIONS: In summary, our data indicate that cryo transfusion restores key fibrinolytic regulators and limits plasmin generation to form stronger clots in an ex vivo laboratory study. This is the first study to investigate differences in clot stability and structure between cryo and Fg-C and demonstrates that the additional factors in cryo allow formation of a stronger and more stable clot.

COVID-19 is a systemic vascular hemopathy: insight for mechanistic and clinical aspects.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury. Severe forms of SARS-CoV-2 infection induce acute respiratory distress syndrome (ARDS) and there is still an ongoing debate on whether COVID-19 ARDS and its perfusion defect differs from ARDS induced by other causes. Beside pro-inflammatory cytokines (such as interleukin-1 ß [IL-1ß] or IL-6), several main pathological phenomena have been seen because of endothelial cell (EC) dysfunction: hypercoagulation reflected by fibrin degradation products called D-dimers, micro- and macrothrombosis and pathological angiogenesis. Direct endothelial infection by SARS-CoV-2 is not likely to occur and ACE-2 expression by EC is a matter of debate. Indeed, endothelial damage reported in severely ill patients with COVID-19 could be more likely secondary to infection of neighboring cells and/or a consequence of inflammation. Endotheliopathy could give rise to hypercoagulation by alteration in the levels of different factors such as von Willebrand factor. Other than thrombotic events, pathological angiogenesis is among the recent findings. Overexpression of different proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) or placental growth factors (PlGF) have been found in plasma or lung biopsies of COVID-19 patients. Finally, SARS-CoV-2 infection induces an emergency myelopoiesis associated to deregulated immunity and mobilization of endothelial progenitor cells, leading to features of acquired hematological malignancies or cardiovascular disease, which are discussed in this review. Altogether, this review will try to elucidate the pathophysiology of thrombotic complications, pathological angiogenesis and EC dysfunction, allowing better insight in new targets and antithrombotic protocols to better address vascular system dysfunction. Since treating SARS-CoV-2 infection and its potential long-term effects involves targeting the vascular compartment and/or mobilization of immature immune cells, we propose to define COVID-19 and its complications as a systemic vascular acquired hemopathy.

Characterisation of a novel thrombomodulin c.1487delC,p.(Pro496Argfs*10) variant and evaluation of therapeutic strategies to manage the rare bleeding phenotype.

INTRODUCTION: A novel variant in the thrombomodulin (TM) gene, c.1487delC,p.(Pro496Argfs*10), referred to as Pro496Argfs*10, was identified in a family with an unexplained bleeding disorder. The Pro496Argfs*10 variant results in loss of the transmembrane and intracellular segments of TM and is associated with an increase in soluble TM (sTM) in the plasma. The aim of this study was to characterise the effect of elevated sTM on thrombin generation (TG) and fibrinolysis, and to evaluate therapeutic strategies to manage the patients. METHODS: Plasma samples were obtained from two patients carrying the variant. TG was triggered using 5 pM tissue factor and measured using the Calibrated Automated Thrombogram. A turbidity clot lysis assay was used to monitor fibrinolysis. TM antigen was quantified by ELISA. RESULTS: Patients with the Pro496Argfs*10 variant had significantly elevated plasma sTM compared to controls (372.6 vs. 6.0 ng/ml). TG potential was significantly lower in patients but was restored by inhibition of activated protein C (APC) or addition of activated Factor VII (FVIIa) or platelet concentrates. In vitro experiments suggested that activated prothrombin complex concentrates (APCC) posed a risk of thrombosis. The time to 50% lysis was significantly prolonged in patients compared to controls, 69.7 vs. 42.3 min. Clot lysis time was shortened by inhibition of activated thrombin activatable fibrinolysis inhibitor (TAFIa). CONCLUSIONS: Our data demonstrate that increased sTM enhances APC generation and reduces TG. Simultaneously, the rate of fibrinolysis is delayed due to increased TAFI activation by sTM. Treatment with platelet or FVIIa concentrates may be beneficial to manage this rare bleeding disorder.

Coagulation status of critically ill patients with and without liver disease assessed using a novel thrombin generation analyzer.

The liver synthesizes the majority of pro- and anti-coagulant and fibrinolytic proteins, and during liver dysfunction synthesis of these proteins is reduced. The end point of conventional hemostatic tests, such as the prothrombin time (PT), occurs when only 5% of thrombin generation (TG) has taken place and is not sensitive to the effects of natural anti-coagulants. The aim of this study was to determine whether TG in the presence of thrombomodulin (TM) provides more useful information about coagulation potential, in comparison to the PT. Analysis was performed on ST Genesia, a novel TG analyzer from Diagnostica Stago. TG was measured using STG-Thromboscreen, a reagent containing an intermediate concentration of human tissue factor (TF) ± rabbit TM to account for anti-coagulant protein C (PC) activity. Platelet-poor plasma (PPP) samples were from the Intensive Care Study of Coagulopathy-2 (ISOC-2), which recruited patients admitted to critical care with a prolonged PT (3 seconds above the reference range). Despite a prolonged PT, 48.0% and 60.7% of patients in the liver and non-liver groups had TG parameters within the normal range. Addition of TM reduced TG by 34.5% and 41.8% in the liver and non-liver groups, respectively. Interestingly, fresh frozen plasma (FFP) transfusion had no impact on TG. Measurement of TG with addition of TM provides a more informative assessment of coagulation capacity and indicates that hemostasis is balanced in patients with liver disease during critical illness, despite conventional tests suggesting that bleeding risk is increased.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing.

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

Clinical bleeding and thrombin generation in admissions to critical care with prolonged prothrombin time: an exploratory study.

BACKGROUND: Prolongation of prothrombin time (PT) is often recorded in critical illness, but has limited ability to predict risk of bleeding. This exploratory study was aimed at assessing a role for thrombin generation (TG) to predict bleeding. STUDY DESIGN AND METHODS: TG was measured by calibrated automated thrombography in admissions to intensive care with prolonged PT. Bleeding events were recorded up to Day 5 after enrollment and correlated with results of PT ratio (PTR) and variables of TG. RESULTS: A total of 306 patients were recruited. A total of 101 bleeding events developed in 46 patients during the period of observation. Many patients with prolonged PT had endogenous thrombin potential (ETP), which was within the normal range (120/251 patients, 47.8%) or even elevated (8%). Although some patients had a reduction in ETP or peak thrombin, these were present over a wide range of PTR. There was no suggestion by receiver operating characteristic analysis that variables of conventional TG were sensitive at predicting bleeding. No bleeding events were documented in patients defined as ETP high, despite elevated PTR. CONCLUSION: Future studies need to explore a role for alternatives tests of coagulation in critical illness. Development of TG assays is required to positively identify more patients at increased bleeding risk or to exclude a larger number at low risk and how this relates to subgroups, such as patients with liver disease, and the need for prophylactic plasma transfusion.

Drug therapy in anticoagulation: which drug for which patient?

Four non-vitamin K oral anticoagulants (NOACs) are now licensed and available in the UK, offering unprecedented choices in anticoagulant therapy for clinicians and patients. NOACs have many clear benefits over warfarin, the most striking being the reduction in intracranial haemorrhage. However, a number of uncertainties remain: their efficacy in certain situations, utility of drug assays, significance of drug interactions and management of bleeding. In the absence of any direct comparative trials, it is not clear that any of the NOACs is significantly better than the others in any of the licensed indications. The differential activities, pharmacokinetics, metabolism, excretion and side effects of the agents should be considered when selecting the most appropriate anticoagulant. In this article, we discuss how, with careful selection for the relevant indication, NOACs can simplify therapy while improving outcomes. We aim to provide clinicians with the information needed to select the most suitable anticoagulant drug for an individual patient in a given situation.

Von Willebrand factor, angiodysplasia and angiogenesis.

The large multimeric glycoprotein Von Willebrand factor (VWF) is best known for its role in haemostasis; however in recent years other functions of VWF have been identified, indicating that this protein is involved in multiple vascular processes. We recently described a new role for VWF in controlling angiogenesis, which may have significant clinical implications for patients with Von Willebrand disease (VWD), a genetic or acquired condition caused by the deficiency or dysfunction of VWF. VWD can be associated with angiodysplasia, a condition of degenerative blood vessels often present in the gastrointestinal tract, linked to dysregulated angiogenesis. Angiodysplasia can cause severe intractable bleeding, often refractory to conventional VWD treatments. In this review we summarise the evidence showing that VWF controls angiogenesis, and review the angiogenic pathways which have been implicated in this process. We discuss the possible mechanisms though which VWF regulates angiopoietin-2 (Ang-2) and integrin αvß3, leading to signalling through vascular endothelial growth factor receptor-2 (VEGFR2), one of the most potent activators of angiogenesis. We also review the evidence that links VWF with angiodysplasia, and how the newly identified function of VWF in controlling angiogenesis may pave the way for the development of novel therapies for the treatment of angiodysplasia in congenital VWD and in acquired conditions such as Heyde syndrome.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells.

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

Mapping the N-glycome of human von Willebrand factor.

vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is ample evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of individuals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn(2635) site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.

Endothelial von Willebrand factor regulates angiogenesis.

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvß3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

Expression of terminal alpha2-6-linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13.

von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by alpha2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (+/- 14%) by ADAMTS13, compared with 11% (+/- 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O > or = B > A > or = AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.

Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor.

Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.

A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF.

ADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (K(D) approximately 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

N-linked glycosylation of VWF modulates its interaction with ADAMTS13.

We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.

Use of recombinant activated factor VII for bleeding in pancreatitis: a case series.

OBJECTIVES: To describe the effects of recombinant activated factor VII (rFVIIa) in the treatment of bleeding in a series of patients with acute or chronic pancreatitis. METHODS: Twelve patients (age, 2.5-65 years) with pancreatitis and bleeding were treated with 18.5 to 120 microg/kg of rFVIIa. Eight patients also had sepsis/infection and/or disseminated intravascular coagulation (DIC). The effects of rFVIIa on bleeding, coagulation status, and transfusion requirements were noted. RESULTS: Bleeding stopped in 4 patients, was markedly reduced in 4 patients, was reduced in 3 patients, and was remained unchanged in 1 patient. For most patients with pre- and post-rFVIIa data, coagulation parameters improved and transfusion requirements reduced. No thrombotic adverse events occurred. Seven patients died for reasons considered to be unrelated to rFVIIa treatment. CONCLUSIONS: This case series indicates that rFVIIa may be an effective hemostatic treatment of patients with pancreatitis suffering from massive bleeding. There were no thromboembolic events in any patient, including those with sepsis or DIC.