Objective: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to... Show moreObjective: The Australian snake venom ptFV (Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa (mammalian factor Va). Approach and Results: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. Conclusions: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity. Show less
A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is... Show moreA wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma. Show less
The direct oral anticoagulants targeting coagulation factor Xa or thrombin are widelyused as alternatives to vitamin K antagonists in the management of venous thromboembolismand nonvalvular atrial... Show moreThe direct oral anticoagulants targeting coagulation factor Xa or thrombin are widelyused as alternatives to vitamin K antagonists in the management of venous thromboembolismand nonvalvular atrial fibrillation. In case of bleeding or emergency surgery,reversal agents are helpful to counteract the anticoagulant therapy and restorehemostasis. While idarucizumab has been established as an antidote for the directthrombin inhibitor dabigatran, reversal strategies for the direct factor Xa inhibitorshave been a focal point in clinical care over the past years. In the absence of specificreversal agents, the off-label use of (activated) prothrombin complex concentrate andrecombinant factor VIIa have been suggested as effective treatment options duringinhibitor-induced bleeding complications. Meanwhile, several specific reversal agentshave been developed. In this review, an overview of the current state of nonspecific andspecific reversal agents for the direct factor Xa inhibitors is provided, focusing on thebiochemistry and mechanism of action and the preclinical assessment of newlyemerging therapies. Show less
Toorop, M.M.A.; Rein, N. van; Cannegieter, S.C.; Meer, F.J.M. van der; Reitsma, P.H.; Lijfering, W.M.; Bos, M.H.A. 2020
Background Major bleeding occurs in 1 to 3% of patients treated with oral anticoagulants per year. Biomarkers may help to identify high-risk patients. A proposed marker for major bleeding while... Show moreBackground Major bleeding occurs in 1 to 3% of patients treated with oral anticoagulants per year. Biomarkers may help to identify high-risk patients. A proposed marker for major bleeding while using anticoagulants is soluble thrombomodulin (sTM). Methods Plasma was available from 16,570 patients of the BLEEDS cohort that consisted of patients who started treatment with vitamin K antagonists between 2012 and 2014. A case-cohort study was performed including all patients with a major bleed (n = 326) during follow-up and a random sample of individuals selected at baseline (n = 652). Plasma sTM levels were measured and stratified by percentiles. Patients were also categorized by international normalized ratio (INR). Adjusted hazard ratios (for age, sex, hypertension, and diabetes) with 95% confidence intervals (CIs) were estimated by means of Cox regression. Results Plasma sTM levels were available for 263 patients with a major bleed and 538 control subjects. sTM levels were dose-dependently associated with risk of major bleeding, with a 1.9-fold increased risk (95% CI: 1.1-3.1) for levels above the 85th percentile versus the <25th percentile. A high INR (>= 4) in the presence of high (>= 70th percentile) sTM levels was associated with a 7.1-fold (95% CI: 4.1-12.3) increased risk of major bleeding, corresponding with a bleeding rate of 14.1 per 100 patient-years. Conclusion High sTM levels at the start of treatment are associated with major bleeding during vitamin K antagonist treatment, particularly in the presence of a high INR. Show less
The venom of the Australian snakePseudonaja textiliscomprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed... Show moreThe venom of the Australian snakePseudonaja textiliscomprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that ofP. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa-factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor-factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile(16)-Asp(194)salt bridge via modification of the activation peptide. Show less
Thrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on... Show moreThrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on Thrombosis assembled basic, translational, and clinical scientists to discuss the origin and potential consequences of thrombo-inflammation in the etiology, diagnostics, and management of patients with cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. This article presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following topics: (1) challenges of the endothelial cell barrier; (2) circulating cells and thrombo-inflammation, focused on platelets, neutrophils, and neutrophil extracellular traps; (3) procoagulant mechanisms; (4) arterial vascular changes in atherogenesis; attenuating atherosclerosis and ischemia/reperfusion injury; (5) management of patients with arterial vascular disease; and (6) pathogenesis of venous thrombosis and late consequences of venous thromboembolism. Show less
The surge in the number of authors per article in the biomedical field makes it difficult to quantify the contribution of individual authors. Conventional citation metrics are typically based on... Show moreThe surge in the number of authors per article in the biomedical field makes it difficult to quantify the contribution of individual authors. Conventional citation metrics are typically based on the number of publications and the number of citations generated by a scientist, thereby disregarding the contribution of co-authors. Previously we developed the p-index that estimates the dependency of a scientist on co-authors during their career. In this study we aimed to evaluate the ability of the p-index to identify researchers with a relatively high degree of scientific dependence on co-authors. For this purpose, we retrieved articles, which were rejected for publication in Journal of Thrombosis and Haemostasis and subsequently published elsewhere. Assuming that authors who were added to a later version of these articles would not fulfill the full authorship criteria, we tested whether these authors showed a larger dependency on co-authors during their scientific career as would be evident from a higher p-index. In accordance with this hypothesis, authors who were added on later versions of articles showed a higher p-index than their peers, indicating an enduring pattern of dependency on other co-authors for publishing their work. This study underscores that questionable authorship practices are endemic to the biomedical research, which calls for alternative methods to evaluate a scientist's qualities. Show less
The venom of the Australian snake Pseudonaja textilis comprises powerful prothrombinactivators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrateliver-expressed... Show moreThe venom of the Australian snake Pseudonaja textilis comprises powerful prothrombinactivators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrateliver-expressed factor X (FX) homologs, including that of P. textilis, comprise anactivation peptide of approximately 45 to 65 residues, the activation peptide of vptFXis significantly shortened to 27 residues. In this study, we demonstrate thatexchanging the human FX activation peptide for the snake venom ortholog impedesproteolytic cleavage by the intrinsic factor VIIIa–factor IXa tenase complex. Furthermore,our findings indicate that the human FX activation peptide comprises anessential binding site for the intrinsic tenase complex. Conversely, incorporation ofFX into the extrinsic tissue factor–factor VIIa tenase complex is completely dependenton exosite-mediated interactions. Remarkably, the shortened activation peptide allowsfor factor V-dependent prothrombin conversion while in the zymogen state. Thisindicates that the active site of FX molecules comprising the v-ptFX activation peptidepartially matures upon assembly into a premature prothrombinase complex. Takentogether, the shortened activation peptide is one of the remarkable characteristics of vptFXthat has been modified from its original form, thereby transforming FX into apowerful procoagulant protein. Moreover, these results shed new light on thestructural requirements for serine protease activation and indicate that catalyticactivity can be obtained without formation of the characteristic Ile16–Asp194 saltbridge via modification of the activation peptide. Show less
Blood coagulation factor Va serves an indispensable role in hemostasis as cofactor for the serine protease factor Xa. In the presence of an anionic phospholipid membrane and calcium ions, factors... Show moreBlood coagulation factor Va serves an indispensable role in hemostasis as cofactor for the serine protease factor Xa. In the presence of an anionic phospholipid membrane and calcium ions, factors Va and Xa assemble into the prothrombinase complex. Following formation of the ternary complex with the macromolecular zymogen substrate prothrombin, the latter is rapidly converted into thrombin, the key regulatory enzyme of coagulation. Over the years, multiple binding sites have been identified in factor Va that play a role in the interaction of the cofactor with factor Xa, prothrombin, or the anionic phospholipid membrane surface. In this review, an overview of the currently available information on these interactive sites in factor Va is provided, and data from biochemical approaches and 3D structural protein complex models are discussed. The structural models have been generated in recent years and provide novel insights into the molecular requirements for assembly of both the prothrombinase and the ternary prothrombinase-prothrombin complexes. Integrated knowledge of functionally important regions in factor Va will allow for a better understanding of factor Va cofactor activity. Show less
Heestermans, M.; Salloum-Asfar, S.; Streef, T.; Laghmani, E.; Salvatori, D.; Luken, B.M.; ... ; Vlijmen, B.J.M. van 2019
Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in... Show moreTissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibodymediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus. Show less
Heestermans, M.; Jong, A. de; Tilburg, S. van; Reitsma, P.H.; Versteeg, H.H.; Spronk, H.M.; Vlijmen, B.J.M. van 2019
Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe(-/-)) mice. The... Show more Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe(-/-)) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe(-/-) mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe(-/-) mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe(-/-) mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.. Show less
Heestermans, M.; Ouweneel, A.B.; Hassan, J.; Kloosterman, M.; Reitsma, P.H.; Gijbels, M.J.J.; ... ; Eck, M. van 2018
