Factor XIII Research Articles

Oral FXIIa inhibitor KV998086 suppresses FXIIa and single chain FXII mediated kallikrein kinin system activation.

Background: The kallikrein kinin system (KKS) is an established pharmacological target for the treatment and prevention of attacks in hereditary angioedema (HAE). Proteolytic activities of FXIIa and single-chain Factor XII (FXII) zymogen contribute to KKS activation and thereby may play roles in both initiating and propagating HAE attacks. In this report, we investigated the effects of potent small molecule FXIIa inhibitors on FXIIa and single chain FXII enzymatic activities, KKS activation, and angioedema in mice. Methods: We examined the effects of 29 structurally distinct FXIIa inhibitors on enzymatic activities of FXIIa and a mutant single chain FXII with R334A, R343A and R353A substitutions (rFXII-T), that does not undergo zymogen conversion to FXIIa, using kinetic fluorogenic substrate assays. We examined the effects of a representative FXIIa inhibitor, KV998086, on KKS activation and both carrageenan- and captopril-induced angioedema in mice. Results: FXIIa inhibitors designed to target its catalytic domain also potently inhibited the enzymatic activity of rFXII-T and the pIC50s of these compounds linearly correlated for rFXIIa and rFXII-T (R 2 = 0.93). KV998086, a potent oral FXIIa inhibitor (IC50 = 7.2nM) inhibited dextran sulfate (DXS)-stimulated generation of plasma kallikrein and FXIIa, and the cleavage of high molecular weight kininogen (HK) in human plasma. KV998086 also inhibited rFXII-T mediated HK cleavage (p < 0.005) in plasma from FXII knockout mice supplemented with rFXII-T and stimulated with polyphosphate or DXS. Orally administered KV998086 protected mice from 1) captopril-induced Evans blue leakage in colon and laryngotracheal tissues and 2) blocked carrageenan-induced plasma HK consumption and paw edema. Conclusion: These findings show that small molecule FXIIa inhibitors, designed to target its active site, also inhibit the enzymatic activity of FXII zymogen. Combined inhibition of FXII zymogen and FXIIa may thereby suppress both the initiation and amplification of KKS activation that contribute to hereditary angioedema attacks and other FXII-mediated diseases.

The relevance of prothrombotic genetic variants in women who experienced pregnancy loss or embryo implantation failure: A retrospective analysis of 1922 cases.

The aim of our study was that to assess the allelic and genotype frequencies of nine prothrombotic gene variants in patients with a history of pregnancy loss and recurrent pregnancy loss (RPL). Women who underwent assisted reproductive technology (ART) with ongoing pregnancy and those with recurrent implantation failure (RIF) were also included. Nine prothrombotic gene variants were evaluated: factor V Leiden (FVL), factor V, H1299R variant (FVR2), factor II (FII) G20210A, methylene-tetrahydrofolate reductase (MTHFR) C677T and A1298C, beta-fibrinogen -455G>A, factor XIII (FXIII) V34L, human platelet antigen-1 (HPA-1) L33P variants, and plasminogen activator inhibitor-1 (PAI-1) 4G/5G. The following study groups were assessed: (1) women who experienced one (n = 334) or two (n = 264) episodes of pregnancy loss; (2) 468 women who experienced RPL; (3) 214 women who underwent ART followed by ongoing pregnancies; and (4) 282 women who experienced RIF after ART, that is, three or more consecutive implantation failures following high-quality embryo transfers to the uterus with an appropriate endometrium. As control group, 430 subjects from the general population were enrolled. FVL, the -455G>A variant of beta-fibrinogen, and PAI-1 4G were associated with a higher risk of developing RPL compared with the general population. Furthermore, FVL, FVR2, FII G20210A and MTHFR C677T conferred a significantly higher risk of RIF in women who performed ART compared with the general population. No statistical differences between the general population and other study groups were observed. Specific prothrombotic genetic variants are more frequently expressed in women with RPL and RIF, supporting their role in the development of polimicrothrombosis and impairing the invasion during embryo implantation.

Low factor XIII levels and altered fibrinolysis in patients with multiple myeloma

BackgroundAcquired factor FXIII (FXIII) deficiency can be immune- or non-immune mediated and may cause severe bleeding symptoms. The incidence of acquired FXIII deficiency and its etiology in patients with multiple myeloma (MM) are poorly understood. ObjectivesTo assess FXIII levels and the balance of fibrinolysis in newly diagnosed, untreated MM and monoclonal gammopathy of undetermined significance (MGUS) patients. MethodsFXIII activity, mixing studies, FXIII-A2B2 antigen, total FXIII-B antigen were measured in platelet-poor plasma from 17 untreated MM patients, 33 untreated MGUS patients, and 30 age and sex-matched healthy controls. Besides routine laboratory measurements, the balance of coagulation and fibrinolysis was evaluated using quantitative fibrin monomer (FM) test, thrombin-antithrombin assay, α2-antiplasmin activity, plasmin-α2-antiplasmin (PAP) complex, D-dimer, plasmin generation assay, clot lysis assay, and ClotPro-TPA test. ResultsFXIII-A2B2 levels were significantly lower in MM patients compared to controls [median (IQR):14.6 (11.2–19.4) vs. 21.8 (17.1–26.4) mg/L, p = 0.0015], whereas total FXIII-B did not differ between groups. Decrease in FXIII activity was parallel to the decrease in FXIII-A2B2. An immune-mediated inhibitory mechanism was ruled out. Free/total FXIII-B was significantly higher in MM patients compared to MGUS and healthy controls, suggesting an etiology of FXIII-A consumption. In MM and MGUS patients, FM, D-dimer, and PAP complex were significantly elevated compared to controls, indicating hypercoagulability and ongoing fibrinolysis. ConclusionsLow FXIII levels due to consumption were observed in MM patients at diagnosis. Hypercoagulability and ongoing fibrinolysis were detected in MM and MGUS, indicating that a disturbed hemostasis balance is already present in the latter benign condition.

Analysis of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ deficiency due to compound heterozygous variants of F12 gene

To analyze the clinical phenotypes and genetic variants of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. A pedigree presented at the First Affiliated Hospital of Air Force Medical University on December 24,2021 was selected as the study subject. Activated partial thromboplastin time (APTT) and coagulation factor Ⅻ activity (FⅫ:C) were determine by a clotting method, and FⅫ antigen was detected with an ELISA assay. Following the extraction of genomic DNA, all exons and flanking regions of the F12 gene were subjected to Sanger sequencing. Clustalx-2.1-win, PROVEAN and Swiss-PDB Viewer software was used to analyze the conservation of amino acids at the variant sites, impact of of the variants on the amino acid substitutions and the protein structure. The APTT of the proband has prolonged to 70.2 s. Her FⅫ:C and FⅫ:Ag have decreased to 12% and 13%, respectively. DNA sequencing revealed that the proband has harbored c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) heterozygous compound missense variants in exons 5 and 13 of the F12 gene, respectively. Her father and sister were heterozygous carriers for the c.346G>A (p.Gly97Ser) variant, whilst her mother and brother were heterozygous for the c.1583C>A (p.Ser509Tyr) variant. The c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) compound heterozygous variants of the F12 gene probably underlay the pathogenesis of hereditary coagulation FⅫ deficiency in this pedigree.

Hemostatic capability of ultrafiltrated fresh frozen plasma compared to cryoprecipitate

This in vitro study evaluated the potential hemostatic effect of fresh frozen plasma (FFP) ultrafiltration on clotting factors, coagulation parameters, and plasma properties. ABO-specific units of FFP (n = 40) were prepared for the concentrated FFP and cryoprecipitate. Plasma water was removed from FFP by ultrafiltration using a dialyzer with a pump running at a 300 mL/min. The aliquot of each concentrated FFP after 50, 100, 200, and 250 mL of fluid removal were measured the standard coagulation assay, clotting activity, and plasma properties to compare those parameters of cryoprecipitate. Concentrated FFP contained 36.5% of fibrinogen in FFP with a mean concentration of 7.2 g/L, lower than the cryoprecipitate level. The levels of factor VIII (FVIII), von Willebrand factor (VWF):antigen (Ag), and VWF:ristocetin cofactor (RCo) were also lower in concentrated FFP, whereas the levels of factor V, factor IX, factor XIII, antithrombin and albumin was higher in concentrated FFP. Maximum clot firmness (MCF) in thromboelastometry was approximately one-half of that in cryoprecipitate. Although the levels of VWF:Ag, VWF:RCo, and FVIII differed depending on the ABO blood types, fibrinogen levels, and MCF were not significantly different among the ABO blood groups in FFP and concentrated FFP.

Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab prophylaxis led us to employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We therefore analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab than in the presence of rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab than as for rFVIII/rFVIIIFc. Scanning electron microscopy and immunofluorescent spinning-disk confocal microscopy of in vivo-generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFcand emicizumab-induced clots. Contrary to the regular fibrin mesh obtained with rFVIII/rFVIIIFc, fibrin fibers appeared to be fused into large patches upon emicizumab treatment. Moreover, fewer red blood cells were detected in regions in which these fibrin patches were present. The presence of highly dense fibrin structures associated with a diffuse fiber structure in emicizumab-induced clots was also observed when using super-resolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

Women have less progression of paroxysmal atrial fibrillation: data from the RACE V study

BackgroundSex differences in atrial fibrillation (AF) are observed in terms of comorbidities, symptoms, therapies received, AF progression and cardiovascular complications.MethodsWe assessed the differences in prevalence and the determinants of AF...

First Validated Model of Zymogen Factor XII Provides Insights into Protein Assembly and Activation

Factor XII (FXII), the zymogen of the plasma protease FXIIa, contributes to thrombo-inflammatory processes. As part of the kallikrein-kinin system, FXIIa converts prekallikrein to the protease kallikrein (PKa), which then cleaves high-molecular-weight kininogen (HK) to release the vasoactive peptide bradykinin. In the disorder hereditary angioedema, dysregulation of FXII activation leads to excessive bradykinin production. When blood is exposed to artificial surfaces, such as during cardiopulmonary bypass or ECMO, FXII is rapidly converted to FXIIa by contact activation, and contributes to thrombus formation by activating factor XI. FXII circulates in plasma in a “closed” form that is resistant to activation by PKa and other proteases. Surface-binding “opens” the FXII structure, facilitating activation. Relatively little is known about the structural features that maintain the closed FXII conformation as the full-length protein has been refractory to crystallization. Recently, a crystal structure was reported for part of the non-catalytic portion of FXII. An intriguing feature of the structure is an interaction between the fibronectin type 2 (FN2) and kringle (KNG) domains at opposite ends of the polypeptide that forms a ring-like structure. Functional studies by our group and others indicate these domains are key to maintaining the closed conformation. Here we describe a model for FXII prepared with the program AlphaFold (AF), and its validation using site-directed mutagenesis to disrupt interactions predicted to maintain the closed conformation. The human FXII model in the AF database was modified by removing residues 300-316 from the proline-rich region and by reference to crystal structures of the FXIIa catalytic domain (CD) bound to inhibitors. Molecular graphics and interdomain interaction analyses were performed in ChimeraX. The refined model predicts 15 interdomain bonds involving residues in FN2, KNG, and the CD. Of the 22 amino acids involved in these bonds, 15 are highly conserved in FXII across 25 vertebrate species. We expressed human FXII in which these residues were individually changed to alanine (Table). Alanine replacements for amino acids not predicted to be involved in interdomain interactions were prepared as controls. Four variants (FXII-D253A, FXII-W257A, FXII-W268A and FXII-E502A) were activated in cell culture and one (FXII-R61A) did not express. FXII-R61A, FXII-W257A, and FXII-W268A were not studied further. FXII-D253A was replaced with FXII-D253K. The active site serine of FXII-E502A was replaced with alanine to prevent autocatalysis (FXII-E502A,S544A). In the absence of a surface, FXII-R36A, FXII-E225A, FXII-D253K, and FXII-K346A were activated by PKa (~15 fold) faster than wild type FXII (FXII-WT) in a chromogenic substrate assay, consistent with an open conformation. Using densitometric analyses of stained SDS- polyacrylamide gels, we noted that these four FXII variants and FXII-E502A,S544A were converted to FXIIa by PKa more rapidly than FXII-WT and other variants. FXII-R36A, FXII-E225A, FXII-D253K, and FXII-K346A bound to prekallikrein with higher affinity than FXII-WT in a plate-binding assay, consistent with a change in conformation. When the four variants were added to human plasma in the absence of a surface, they caused a rapid cleavage of HK by accelerating reciprocal activation with prekallikrein. Our functional analyses indicate that non-covalent bonds between R36 on FN2 and E502 on the CD; R36 and E225 on KNG; and D253 on KNG and K346 adjacent to the activation cleavage site are critical for maintaining FXII in a closed conformation. The instability of FXII variants with substitutions for tryptophans at positions 257 and 268 also implicate interactions between these residues and K346. In the AF model these bonds appear to maintain FXII in a conformation in which access to the activation cleavage site is limited. Our binding studies also suggest the closed conformation mask a binding site for prekallikrein and PKa. These bonds are likely disrupted when FXII binds to a surface, as part of the contact activation mechanism that enhances FXII activation.

Forms of Hereditary Angioedema That Are Independent of Kallikrein and Factor XIIa System

Hereditary angioedema (HAE) is an inherited disorder characterized by recurrent episodes of severe soft tissue swelling involving skin and mucous membranes. In most cases, edema is caused by excessive formation of the vasoactive nanopeptide bradykinin (BK) due to dysregulation of the plasma kallikrein-kinin system (KKS). The KKS consists of the zymogens prekallikrein (PK) and factor XII (FXII) and the cofactor/substrate high-molecular-weight kininogen (HK). PK and FXII reciprocally convert each other to the proteases plasma kallikrein (PKa) and FXIIa. PKa cleaves HK after Lys362 and Arg371 to release bradykinin. In most cases of HAE, increased KKS activation is due to low plasma activity of C1-Inhibitor (C1-INH), the main regulator of PKa and FXIIa. However, ~10% of HAE patients have normal C1-INH activity (HAEnC1). A relationship between fibrinolysis and kinin formation was first described in 1971. The fibrinolytic protease plasmin activates FXII and cleaves HK, although it is unclear that this results in significant bradykinin generation. Recently mutations in the PLG gene encoding plasminogen (Lys311Glu) and the KNG1 gene encoding HK and the related protein low-molecular-weight kininogen (LK, Met361Lys) were identified in patients with HAEnC1. Here we describe how the fibrinolytic system contributes to angioedema in patients with these mutations. Human Glu-plasminogen (Plg), HK and LK were expressed in HEK293 cells. Lys311 in plasminogen was changed to glutamic acid, and Met361 in HK and LK were changed to lysine, by site directed mutagenesis. Wild type (Plg-Lys311) and variant (Plg-Glu311) plasminogen were converted to plasmin (Plm-Lys311 and Plm-Glu311) with urokinase. The capacity of plasmin to cleave HK and LK were studied with SDS-PAGE, western blots and kinin ELISA. Cleavage of wild type HK-Met361 and LK-Met361 and variant HK-Lys361 and LK-Lys361 by PKa and plasmin were assessed in a similar manner. The types of kinin released during reactions were determined by mass spectroscopy. Normal plasma was supplemented with Plg-Lys311 or Plg-Glu311, and tPA was added to generate plasmin. Bradykinin release was substantially greater with Plm-Glu311 than Plm-Lys311. This held when reactions were run in plasma lacking FXII or PK, indicating activation of the KKS was not responsible for kinin production. Using plasma-derived proteins, PKa released bradykinin 100-fold faster from HK than LK, and released bradykinin from HK ~50-fold faster than did plasmin. PKa and plasmin released bradykinin at comparable relatively slow rates from LK. Plm-Glu311 released bradykinin from HK 10-fold faster, and from LK 3-fold faster, than in reactions with Plm-Lys311. In experiments in which kininogen deficient plasma was supplemented with either HK or LK to a physiologic concentration, the majority of bradykinin released by Plm-Glu311 was from LK. PKa-catalyzed release of bradykinin from wild type HK-Met361 and variant HK-Lys361 comparably. The same held for LK-Met361 and LK-Lys361. However, plasmin released kinin from HK-Lys361 and LK-Lys361 3.3-fold and 13-fold faster than from HK-Met361 and LK-Met361. While the kinin released from HK-Met361 and LK-Met361 was bradykinin, plasmin released the decapeptide Lys-bradykinin (kallidin) from HK-Lys361 and LK-Lys36. Our studies indicate that angioedema in some patients with HAEnC1 may be due to fibrinolytic activity, and not to KKS activity. Both HK and LK may be substrates in such patients. As the plasma LK concentration is ~4-fold higher than HK, it may be the main pathologic kinin source. The plasmin Lys311Glu substitution converts the protease into a more efficient kininogenase. Fibrinolytic activity is normally brisk in the mouth, perhaps explaining why patients with the Lys311Glu mutation primarily suffer from oral-lingual angioedema. The findings raise the possibility that oral-lingual edema after tPA infusion or ACE inhibitor administration may also be due to plasmin-mediated cleavage of LK and HK. The Met361Lys substitution in HK and LK introduces a novel protease cleavage site immediately upstream from a normal PKa cleavage site. The novel site is preferred over the normal site by plasmin, but not PKa. Several treatments for HAE directed at neutralizing PKa or FXIIa activity have been developed. Our results suggest that such drugs may be less effective in patients with HAEnC1 in which disease mechanisms operate independently of the KKS.

Transferrin and Erythropoietin Increased Levels Correlate with Thrombosis at High Altitude

Living at high altitude involves great interest because of the challenging multiple adaptive responses to a hypoxic environment. Native Bolivian Andeans have lived at an altitude of 4000 m in The Andes mountains for about 14,000 years exposed to a low atmospheric pressure (453 mmHg) and low oxygen concentration (12,7%). It was described Andeans adaptation to high altitude is mainly related to cardiovascular issues (PMID: 29100088), with a low significantly genetic selection in HIF pathway components responsible of erythropoiesis regulation (PMID: 36980912), so that they have adapted to exist with increased hemoglobin concentrations. Thus, Andeans can be prevalent to undergone hematological and thromboembolic disorders. Thrombotic events have been reported to be increased at high altitude, and this recently associated to increased transferrin (Tf) (PMID: 36040436), likewise to increased erythropoietin (Epo) (Amaru, RevMed 2022). Increased Tf were described to be induced by both iron deficiency and hypoxia via HIF (PMID: 9242677). In addition, iron deficiency was postulated to inhibit the function of prolyl hydroxylase 2 (PHD2) in the hydroxylation of HIF-2, necessary for recognition and degradation by VHL (PMID:22304911). Under normal conditions, Tf is bound to fibrinogen at a molar ratio of 4:1, leading to plasma Tf sequestration by fibrinogen and leaving little Tf free in the circulation. While at hypoxia conditions, abnormally upregulated Tf potentiates thrombin, factor XIIa and inhibits antithrombin, inducing hypercoagulability (PMID:36040436). Similarly, increased HIF1a at high altitude increases Tf promoting the transferrin gene expression, which contains HIF-1α binding sites in its enhancer region (PMID:36844187). HIF is a DNA-binding transcription factor for activating expression of Epo gene (PMID:32561149). Epo, in turn, is increased due to the hyperegulation of HIF as expected at low atmospheric pressure environments. Epo activates the hypoxia-inducible factor that leads to Epo synthesis to stimulate red blood cell production, which also increases the demand for iron to synthesize hemoglobin (PMID:21078592). This, added to a decreased iron and increased HIF 1a, gives rise to a hyperregulation of Tf. Epo has been described to have prothrombotic properties and be related to thrombotic events (PMID:10779449; Amaru, RevMed 2022). In this sense, to further observe a correlation of transferrin and erythropoietin increased levels with the risk of thrombotic events at high altitude, we studied patients with erythrocytosis, anemia and polycythemia vera. We analyzed clinical, lab tests and epidemiological data of 920 patients with Chronic Mountain Sickness erythrocytosis (CMS-e) (n=560), anemia (n=372), and Polycythemia Vera (PV) (n=20), all Bolivian Andeans born and residing at 4000 m. Considering at this altitude normal hemoglobin concentrations vary from 14-17 g/dl in women and 15-18 g/dl in men, erythrocytosis condition comprises Hb levels &amp;gt;18 g/dl in women and &amp;gt;19 g/dl in men (Amaru et al, Rev Hematol Mex, 2016); similarly, it embraces Hb &amp;lt;12 g/dl for women and &amp;lt;13 g/dl for men in anemias (PMID: 23317073). Positive JAK F617V tests were corroborated in PV patients. Iron deficiency correlation considered serum ferritin &amp;lt;30 ug/L and MCV &amp;lt;80 fL (Clark et al, Nutrition, 2008). Eco-doppler study records confirmed the occurrence of thrombotic events. Data analysis was performed through Excel 16.29.1 and SPSS program, Chi-squared, Fishers and Pearson tests were done. We observed significative increased Tf levels in erythrocytosis, anemia, and PV patients mainly on those presenting iron levels deficiency. Epo was highly increased in erythrocytosis patients with iron deficiency. These results correlated with the incidence of thrombotic events in erythrocytosis (CMS-e) and anemia patients with iron deficiency. Age was also relevant in both groups. (Table 1). The increase of Epo levels correlated with thrombosis in erythrocytosis and PV patients with iron deficiency (Table 1). Transferrin and Erythropoietin increased levels at high altitude correlated with thrombosis in Chronic Mountain Sickness erythrocytosis and iron deficiency anemia patients. However, Increased Tf did not correlate with thrombosis in PV patients at high altitude. Our data confirm that increased Tf and Epo have prothrombogenic properties in CMS-e, iron deficiency anemia patients at high altitude.

Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) and Cryoprecipitated AHF Contain Von Willebrand Factor with Comparable Binding to Collagen and Support Shear-Induced Platelet Thrombus Formation

Traumatic hemorrhage is a leading cause of morbidity and mortality and is a potentially preventable cause of death. Trauma-induced coagulopathy (TIC) exhibits complex pathophysiology. Fibrinogen (FIB) deficiency, a major mediator of TIC, is an independent predictor of mortality; and one of the first factors to reach critically low levels during hemorrhage. Early supplementation of FIB using cryoprecipitated AHF (Cryo) administration in resuscitation has been shown to improve outcomes in the treatment of TIC. Unlike FIB concentrates, Cryo prepared from conventional plasma is enriched in von Willebrand factor (VWF), factor VIII, factor XIII, and fibronectin that are impacted by TIC in addition to FIB. However, Cryo has a limited post thaw shelf-life of 4-6 hours at room temperature. This causes logistical impediments for early clinical application in trauma often resulting in use late in resuscitation with significant wastage. Pooling of individual Cryo units to achieve therapeutic doses increases donor exposure with risk of bacterial and viral transfusion transmitted infections (TTI). FDA approved Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) manufactured from Amotosalen/UVA treated plasma (PR plasma) is an alternative FIB source that has a 5-day post-thaw shelf-life at RT with reduced TTI risk. Due to its longer post-thaw shelf-life, it can be thawed in advance so that it is readily available and can be administered early in massive transfusion protocols with significantly lower wastage. VWF is a key component of Cryo and plays a major role in hemostasis during resuscitation with TIC. Here we have compared VWF contained in Cryo and IFC in various functional assays. At equimolar concentrations as assessed by measured VWF antigen, IFC and Cryo supported similar ristocetin-induced platelet aggregation. Accordingly, VWF multimeric composition, particularly presence of the largest molecular species, was similar in IFC and Cryo. We then evaluated the ability of VWF in IFC and Cryo compared to native blood to support platelet adhesion and platelet-rich thrombus formation onto a fibrillar type I collagen surface exposed to a linearly increasing shear rate gradient (0 t0 20,000 s -1) in the direction of flow from inlet to outlet of a Hele-Shaw flow chamber. Briefly, human blood, collected in EDTA, Lepirudin and supplemented with PGE 1 was centrifuged to separate blood cells from plasma. Red cells and platelets were washed to remove all plasma VWF and reconstituted to original hematocrit with Tyrode buffer at pH 7.4 spiked with either Cryo or IFC up to 1 U/ml of VWF. Platelets in native blood and in the different mixtures were fluorescently labeled with 3,3'-dihexyloxacarbocyanine iodide (DiOC 6) for visualization, and image sequences were acquired at different shear rates along the gradient. Images were analyzed to quantify platelet adhesion (the proportion of the field of view covered by platelets) and aggregation (i.e., the number and area of aggregates) at any given shear rate. As a negative control, blood reconstituted with buffer instead of a VWF source was used to ascertain that plasma VWF was effectively removed. As positive control, normal donor blood was used without washing out VWF (native blood). At all tested shear rates, the total surface coverage (1 pixel = 0.099 µm 2) by platelets was comparable with VWF in AHF or IFC added to washed blood and native blood (Figure). Of note, the individual size of platelet aggregates tended to increase with increasing shear rates and the number of individual aggregates tended to decrease. Specific monoclonal antibodies against VWF A1 and A3 domains - blocking interaction with GPIbα and collagen, respectively - markedly reduced the number and size of platelet aggregates over the surface. These results indicate that VWF derived from IFC, comparable to Cryo and native blood, retains functions strictly required to support platelet adhesion and aggregation at extremely elevated levels of shear rate/stress. The figure shows the size where 1 pixel = 0.099 µm 2 of each individual platelet thrombus on the surface.

Management of Immune Thrombotic Thrombocytopenic Purpura without Therapeutic Plasma Exchange: Analysis of Efficacy and Safety Data

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening autoimmune disorder caused by ADAMTS13 deficiency. Caplacizumab, an anti-VWF nanobody, is approved for iTTP treatment, reducing the need for therapeutic plasma exchange (TPE) and improving platelet recovery and survival. Methods: We conducted a retrospective study on 42 acute iTTP cases in Austria and Germany, treated with a modified regimen aimed at avoiding TPE if platelet count increased post-first caplacizumab dose. An extensive efficacy and safety analysis of this approach was performed, in comparison with a control group of 59 patients with iTTP, receiving frontline caplacizumab treatment with TPE and immunosuppression (standard of care, SOC). The main outcome was time to platelet count normalization. Secondary outcomes included clinical response, exacerbation, refractory iTTP, and iTTP-related deaths. Retrospective safety assessments with evaluation of treatment emergent adverse events (TEAE) were performed in both cohorts. Results: The median time to platelet count normalization was similar between the two cohorts (3 and 4 days; P = 0.31, see Figure 1). There were no significant differences in clinical response, exacerbations, refractoriness, or iTTP-related deaths (see Table 1). Four patients did not respond to the first dose of caplacizumab, and TPE was initiated. Cytomegalovirus infection, HIV/hepatitis B co-infection, and ovarian teratoma may have hindered immediate treatment response. Platelet count doubling and median platelet count change after first caplacizumab dose were unaffected by TPE. The median time to LDH normalization was 11 days in the TPE-free cohort. During overall follow-up at least one adverse event (TEAE) was observed in 11 patients (26.2%) in the TPE-free group and 15 patients (25.4%) in the SOC group (excluding adverse events of iTTP that were evaluated as outcome parameters) (see Table 1). There was one TTP-related death reported in the SOC group (1.7%). The cause of death in this patient was cerebral ischemia due to multiple strokes. Serious adverse events associated with therapeutic plasma exchange were observed in 3 patients (5.1%) exclusively in the SOC cohort. These included (1) in-hospital cardiac arrest (IHCA), (2) generalized seizure and supraventricular tachycardia and (3) a transfusion reaction with hypotension, rigors, pruritus and exanthema. Bleeding complications were reported without a significant difference and mainly comprised gingival bleeding and epistaxis. Two patients in the TPE-free cohort (4.8%) experienced major bleeding events, namely subdural hematoma requiring surgical intervention and gastrointestinal bleeding with mass transfusion. Spontaneous subdural hematoma occurred in one patient after 20 days of caplacizumab treatment. Gastrointestinal bleeding from a colon diverticulum occurred in another patient after a clinical response and resulted in a significant drop in hemoglobin and thrombocytes. Gastrointestinal bleeding worsened with concomitant factor XIII deficiency, which was reconstituted intravenously in addition to mass transfusion. Conclusion: Caplacizumab and immunosuppression, without TPE, rapidly controlled microvascular thrombosis and achieved a sustained clinical response in iTTP. The efficacy was similar to the control group, which received SOC. Standard iTTP treatment with TPE may lead to a prolonged hospital stay, including admission to the ICU, multiple sessions of daily TPE with associated risks, and the associated costs of plasma, equipment, nursing care, and medical supervision. The number of patients experiencing critical adverse events was comparable between groups. Bearing all restrictions of a retrospective cohort in mind, we did not identify a critical safety signal for TPE-free iTTP-treatment. However, TPE itself resulted in three life-threatening adverse events in the SOC cohort, including an in-hospital cardiac arrest. In conclusion, omitting TPE appears to be a viable choice for acute iTTP management that may reduce treatment burden without compromising patient outcomes or safety.

Neuraxial Analgesia in Pregnant Individuals with Bleeding Disorders: A Comprehensive Examination of Obstetric Anesthesia Practices and Outcomes

Introduction: Neuraxial analgesia in obstetric anesthesia offers multiple advantages when compared with general anesthesia, including better pain relief and patient satisfaction, reduced opioid-related side effects, and decreased maternal morbidity and mortality. However, neuraxial analgesia carries a risk of hematoma, which can lead to permanent neurological complications, and is considered contraindicated in patients with uncorrected bleeding disorders. Hemostatic thresholds for safe neuraxial analgesia remain undetermined. Current evidence for neuraxial analgesia in pregnant individuals with bleeding disorders is limited to a few case reports or small case series, leaving significant gaps in clinical practices based on expert opinions. We sought to examine obstetric analgesia practices and outcomes in pregnant individuals with bleeding disorders. Specifically, we aimed to determine the methods used at the time of labor and delivery, the concordance with antenatal recommendations, and the outcomes in relation to the bleeding disorder, hemostatic thresholds, and hemostatic therapy support. Methods: A retrospective cohort study was conducted on all pregnant individuals with bleeding disorders who delivered between 2010-2020 at our institution, a large tertiary academic care center with high-risk obstetrical care in Ontario, Canada. We collected data on patient and disease characteristics, delivery mode, hemostatic laboratory values, hemostatic support, obstetric analgesia method, antenatal anesthesia and hematology recommendations, and post-procedure outcomes. A descriptive analysis was performed to present the findings. Results: The study cohort comprised of 56 pregnant patients, resulting in 82 deliveries. The median maternal age was 31.5 years (interquartile range (IQR): 28-34), and the median gestational age was 39 weeks (IQR: 38-40). Most patients delivered vaginally (60/82, 73.2%). von Willebrand disease type 1 (VWD-1) (28/82, 34.1%) and hemophilia A (24/82, 29.3%) were the most prevalent bleeding disorders. Neuraxial analgesia was used in most deliveries (59/82, 72.0%), followed by opioids (25/82, 30.5%), and nitrous oxide (15/82, 18.3%). Antenatal neuraxial anesthesia recommendations were documented by anesthesiology in 59.8% (50/82) and by hematology in 82.9% (68/82) of deliveries. The concordance rate between the two specialties was high at 95.7%. In two cases with discordant recommendations, neuraxial anesthesia was performed without hemostatic therapy, and no adverse events were reported. A diagnosis of bleeding disorder was established prior to the pregnancy in 79 deliveries (96.3%). Three patients were diagnosed after pregnancy: one with VWD-1 and baseline von Willebrand factor (VWF) activity at 0.34 IU/mL, one with VWD-1 and baseline VWF activity at 0.44 IU/mL, and one with Factor XIII deficiency and baseline levels at 0.42 IU/mL. They had neuraxial anesthesia administered without bleeding complications. Hemostasis laboratory values in the third trimester were considered adequate for neuraxial analgesia in 68.4% (54/79) of deliveries. Those who did not reach adequate levels received hemostatic therapy support prior to neuraxial analgesia, except for three patients (one with VWD-1 with VWF activity &amp;lt; 0.50 IU/mL, one with VWD type 2 with VWF activity &amp;lt; 0.50 IU/mL, one with factor XI deficiency and levels &amp;lt; 0.40 IU/mL) who fortunately did not develop post-procedural hematomas. We observed no bleeding events following neuraxial analgesia, but there were two adverse events (pain at epidural site) necessitating emergency room visits. Conclusions: Our study represents the largest cohort of pregnant individuals with bleeding disorders receiving neuraxial anesthesia and provides evidence supporting the safe administration of neuraxial analgesia, with high level of adherence to antenatal recommendations. In most cases, hemostatic laboratory values were deemed adequate, obviating the need for additional hemostatic therapy support. These findings contribute to our understanding of obstetric anesthesia practices in this population, thereby facilitating the provision of safe and effective analgesia during labor and delivery. Future prospective and multicenter studies focusing on the obstetrical management of pregnant persons with bleeding disorders, with specific emphasis on neuraxial analgesia, are warranted.

Earliest Possible Detection and Intervention for Synovitis in Hemophilia- Results from a Single Center Retrospective Cohort Study

Introduction There is scientific evidence to suggest that the most serious health problems for hemophilic patients arise from joint damage, which in turn results from joint bleeding, which is overlooked and therefore not treated. The ankle joint was affected the earliest in most hemophilic patients. Strategies for detecting and treating bleeding in hemophilic patients need to be further developed and made more widely known by clinicians. Aim of our study The aim of our retrospective, single-center cohort study was to compare the effects of reactive versus preventive joint ultrasound as guidance for adjustments of treatment regimens for patients with severe hemophilia at our Hemophilia Comprehensive Care Center (HCCC) in Duisburg, Germany. Methods Before 2020, we used ultrasound according to the German hemophilia-synovitis guidelines in 2018. This meant that we sonographed the ankle joints and knees of most of our patients when they reported discomfort in the joint or when a joint bleeding had occurred. In these cases, prophylactic hemophilia treatment was immediately adjusted to a level at which the bleeding and/or discomfort stopped. If the factor treatment was not sufficient to accomplish this goal, the joints were treated with radiosynoviorthesis (RSO). In 2020, we changed our diagnostic strategy from reactive to preventive measures. Thus, we used ultrasound on every ankle and knee joint of our severely hemophilic patients at least once a year. If we found so called “silent symptoms,” which means touch-sensitive joints, the joints were sonographed once every three months. In cases of signs of joint degradation, we adjusted the prophylactic factor treatment accordingly. Results From 2020 to 2022, we examined a total of 1193 joints of 688 patients with hemophilia A or B, as well as with vWD disease type 2 or 3 and other severe coagulation disorders (factor VII deficiency, factor XIII deficiency, afibrinogenemia, and Glanzman disease). Of these, 103 had severe hemophilia A or B. In this study, we examined 656 joints sonographically. Of these, we examined 281 ankle joints in 80 patients and 175 knees in 64 patients, and adjusted the therapy regimens accordingly in these patients if necessary. Seven RSOs of the ankle joints and three RSOs of the knees were performed in the cohort up to 2020. After 2020, only two RSOs of the ankle joints were performed in the cohort. Conclusions At least for patients at our HCCC in Duisburg, Germany, we were able to show for the period 2020-2022 that closer ultrasound monitoring of ankle joints and knees in patients with severe hemophilia A or B and appropriate adjustment of prophylactic factor therapy was associated with less severe and less frequent joint degradation.

Statins Effects on Blood Clotting: A Review.

Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase, which are largely used in patients with or at risk of cardiovascular disease. Available data on thromboembolic disease include primary and secondary prevention as well as bleeding and mortality rates in statin users during anticoagulation for VTE. Experimental studies indicate that statins alter blood clotting at various levels. Statins produce anticoagulant effects via downregulation of tissue factor expression and enhanced endothelial thrombomodulin expression resulting in reduced thrombin generation. Statins impair fibrinogen cleavage and reduce thrombin generation. A reduction of factor V and factor XIII activation has been observed in patients treated with statins. It is postulated that the mechanisms involved are downregulation of factor V and activated factor V, modulation of the protein C pathway and alteration of the tissue factor pathway inhibitor. Clinical and experimental studies have shown that statins exert antiplatelet effects through early and delayed inhibition of platelet activation, adhesion and aggregation. It has been postulated that statin-induced anticoagulant effects can explain, at least partially, a reduction in primary and secondary VTE and death. Evidence supporting the use of statins for prevention of arterial thrombosis-related cardiovascular events is robust, but their role in VTE remains to be further elucidated. In this review, we present biological evidence and experimental data supporting the ability of statins to directly interfere with the clotting system.

Coagulation factor XIII is a critical driver of liver regeneration after partial hepatectomy

BackgroundActivation of coagulation and fibrin deposition in the regenerating liver appears to promote adequate liver regeneration in mice. In humans, perioperative hepatic fibrin deposition is reduced in patients who develop liver dysfunction after partial hepatectomy (PHx), but the mechanism underlying reduced fibrin deposition in these patients is unclear. Methods and ResultsHepatic deposition of cross-linked (ie, stabilized) fibrin was evident in livers of mice after two-thirds PHx. Interestingly, hepatic fibrin cross-linking was dramatically reduced in mice after 90% PHx, an experimental setting of failed liver regeneration, despite similar activation of coagulation after two-thirds or 90% PHx. Likewise, intraoperative activation of coagulation was not reduced in patients who developed liver dysfunction after PHx. Preoperative fibrinogen plasma concentration was not connected to liver dysfunction after PHx in patients. Rather, preoperative and postoperative plasma activity of the transglutaminase coagulation factor (F)XIII, which cross-links fibrin, was lower in patients who developed liver dysfunction than in those who did not. PHx-induced hepatic fibrin cross-linking and hepatic platelet accumulation were significantly reduced in mice lacking the catalytic subunit of FXIII (FXIII−/− mice) after two-thirds PHx. This was coupled with a reduction in both hepatocyte proliferation and liver–to–body weight ratio as well as an apparent reduction in survival after two-thirds PHx in FXIII−/− mice. ConclusionThe results indicate that FXIII is a critical driver of liver regeneration after PHx and suggest that perioperative plasma FXIII activity may predict posthepatectomy liver dysfunction. The results may inform strategies to stabilize proregenerative fibrin during liver resection.

Fibrin and Fibrinolytic Enzyme Cascade in Thrombosis: Unravelling the Role.

Blood clot formation in blood vessels (thrombosis) is a major cause of life-threatening cardiovascular diseases. These clots are formed by αA-, βB-, and ϒ-peptide chains of fibrinogen joined together by isopeptide bonds with the help of blood coagulation factor XIIIa. These clot structures are altered by various factors such as thrombin, platelets, transglutaminase, DNA, histones, and red blood cells. Various factors are used to dissolve the blood clot, such as anticoagulant agents, antiplatelets drugs, fibrinolytic enzymes, and surgical operations. Fibrinolytic enzymes are produced by microorganisms (bacteria, fungi, etc.): streptokinase of Streptococcus hemolyticus, nattokinase of Bacillus subtilis YF 38, bafibrinase of Bacillus sp. AS-S20-I, longolytin of Arthrobotrys longa, versiase of Aspergillus versicolor ZLH-1, etc. They act as a thrombolytic agent by either enhancing the production of plasminogen activators (tissue or urokinase types), which convert inactive plasminogen to active plasmin, or acting as plasmin-like proteins themselves, forming fibrin degradation products which cause normal blood flow again in blood vessels. Fibrinolytic enzymes may be classified in two groups, as serine proteases and metalloproteases, based on their catalytic properties, consisting of a catalytic triad responsible for their fibrinolytic activity having different physiochemical properties (such as molecular weight, pH, and temperature). The analysis of fibrinolysis helps to detect hyperfibrinolysis (menorrhagia, renal failure, etc.) and hypofibrinolysis (diabetes, obesity, etc.) with the help of various fibrinolytic assays such as a fibrin plate assay, fibrin microplate assay, the viscoelastic method, etc. These fibrinolytic activities serve as a key aspect in the recognition of numerous cardiovascular diseases and can be easily produced on a large scale with a short generation time by microbes and are less expensive.

Structural properties of newly 4,7-dihydroxycoumarin derivatives as potential inhibitors of XIIa, Xa, IIa factors of coagulation

Some coumarin derivatives show notable anticoagulant properties through their ability to interrupt the enzymatic function of vitamin K epoxide reductase. The current challenge lies in the development of novel derivatives that are more efficient and less harmful, specifically targeting the direct inhibition of coagulation factors. This study examined the direct inhibitory potential of two recently synthesized coumarin derivatives, 3-(1-(3-hydroxy-4-methoxyphenyl)-amino)-ethyldiene)-2,4-dioxochroman-7-yl acetate (A-3OH) and 3-(1-(4-hydroxy-3-methoxyphenyl)-amino)-ethyldiene)-2,4-dioxochroman-7-yl acetate (A-4OH), according to coagulation factors IIa, Xa, and XIIa. Accurately defining the structural properties of compounds is of great importance for a comprehensive understanding of the biological activity of pharmaceutical agents. In regard, this study aimed to analyze the structural (Potential Energy Surface (PES)), spectroscopic (FT-IR, NMR, and UV–Vis), and electronic properties (Quantum Theory of Atoms in Molecules (QTAIM)) of two newly synthesized compounds. These properties are determined using a combination of experimental and theoretical methods (B3LYP-D3BJ/6–311++G(d,p) level of theory). The investigated compounds deviate from planarity based on the results of structural properties. The results of QTAIM analysis show the presence of partially covalent hydrogen bonds and closed-shell van der Waals interactions that stabilize the examined compounds. Based on the large correlation coefficient (R) and low mean absolute error (MAE) of the spectroscopic data showed that the applied level of theory effectively replicated the experimental finding. The results from the molecular docking study suggest that both compounds demonstrate similar inhibitory effects on IIa when compared to the standard inhibitor dabigatran (DAB). The efficacy of A-3OH in inhibiting Xa is similar to that of conventional inhibitors apixaban (API), and rivaroxaban (RIV). The observed inhibitory effect of both compounds on coagulation factor XIIa was more potent than that of the conventional co-crystallized inhibitor. The results of this study add to the understanding of the structure-activity relationship of coumarin derivatives and provide possibilities for enhancing anticoagulant therapy and clinical outcomes for coagulation-related illnesses. The findings lay a foundation for further in vitro and in vivo investigations into the anticoagulant activities of the compounds, contributing to their potential pharmaceutical application.

Discovery of potential plasma protein biomarkers for ventricular fibrillation due to first ST-elevation myocardial infarction via proteomics

Abstract Background Although most sudden cardiac deaths are a consequence of ventricular fibrillation (VF) subsequent to myocardial infarction, the underlying biological mechanisms of VF in the setting of ischemia are largely unknown. We used an unbiased mass-spectrometry (MS)-based proteomic approach to improve our understanding of the molecular VF mechanisms. Purpose We aimed to identify and characterize proteins that are associated with VF during first ST-elevation myocardial infarction (STEMI). Methods We included 230 participants from a Danish ongoing case-control study on patients with first STEMI with VF (case, n=110) and without VF (control, n=120) before guided catheter insertion for primary percutaneous coronary intervention. Using MS-based proteomics on plasma samples collected within 24h of symptom onset, we investigated the plasma proteome. After quality control, one patient was excluded and the difference in the quantitative proteome composition between cases and controls were analysed using t-tests, and further corrected for multiple comparison using the Benjamini-Hochberg procedure (q-value). Cardiac enzymes and anterior infarct location were used in a subset as a proxy for large myocardial damage. Results In 229 STEMI patients (72% men, median age 62 years (interquartile range (IQR): 54-70)), a total of 401 unique proteins were identified with a median of 257 proteins (IQR: 244-281) quantified per patient. A total of 26 proteins were associated with VF, these proteins were involved in several biological processes. The most frequent processes were blood coagulation, hemostasis and immunity. After correcting for multiple testing, two up-regulated proteins remained significantly associated with VF, actin beta-like 2 (ACTBL2, FC 2.25, p&amp;lt;0.001, q=0.023) and coagulation factor XIII-A (F13A1, FC 1.48, p&amp;lt;0.001, q=0.023). The most up-regulated protein was ACTBL2, and the second most up-regulated protein was actin alpha 2 (ACTA2, FC 1.72, p=0.001, q=0.073). None of the three proteins were correlated with cardiac enzymes or anterior infarct location. Conclusion VF due to first STEMI was significantly associated with two up-regulated proteins (ACTBL2 and F13A1). None of these proteins were correlated with infarct size which could reflect novel underlying molecular VF mechanisms.

Abstract 15197: Differential Impact of Factor XII, Factor XI and Prekallikrein Deficiency on Thrombosis: A Pro-Thrombotic Vascular Action of Bradykinin

Objective: Factor XII (FXII) initiates the plasma contact system that triggers the intrinsic coagulation pathway and the kallikrein-kinin pathway via factor XI (FXI) and plasma prekallikrein (PK), respectively. The importance of the contact system for thrombosis sparing hemostasis is well established, however a comprehensive head-to-head comparison of the three proteases in arterial thrombosis is missing. Approach and Results: We produced mice deficient in FXII ( F12 -/- ), FXI ( F11 -/- ) and PK ( Klkb1 -/- ) and compared them in two distinct thrombosis models. While all (100%) of WT mice formed occlusive thrombi upon 30% ferric chloride challenge, carotid artery occlusion was reduced to 0, 4.8, 27.3 % in F12 -/- , F11 -/- and Klkb1 -/- mice (n=14-22), respectively. Upon photochemical-induced cremaster arteriolar thrombosis, occlusion time (s) prolonged to 226.1±60.4, 123.8±32.0 and 87.8±25.3 (mean±SEM, n=10-15) respectively in F12 -/- , F11 -/- and Klkb1 -/- mice, largely exceeding WT levels (38.3±12.1). A discordance was observed between in vivo thrombotic effects of the proteases and their roles for ex vivo coagulation, which ranked FXII≈FXI&gt;PK as assayed by thromboelastography and activated partial thromboplastin time. Activation of FXII promoted thrombosis in WT and Klkb1 -/- mice, and impressively, it also accelerated thrombosis in F11 -/- mice, increasing activated PK activity and plasma bradykinin formation. Infusion of bradykinin and blockade of degradation of endogenous bradykinin increased platelet-endothelium adhesion and promoted thrombotic occlusions, which involved bradykinin mediated endothelial cell-release of platelet activating factor, rather than direct activation of platelets by bradykinin. Conclusion: FXII, FXI and PK differentially mediate experimental thrombosis in vivo and FXII/FXI appear as superior targets for thrombosis as compared to PK. PK- bradykinin axis activated by FXII contributes to thrombosis in vivo .