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Background: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis., Objectives: To unravel and prevent EGF-1-mediated FXII surface-binding with a variable domain of heavy chain-only antibody (V H H)., Methods: FXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain-specific V H Hs were raised to inhibit EGF-1-mediated FXII contact activation while preserving quiescence., Results: Two unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1-specific V H Hs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V H H "F2" binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence., Conclusion: The 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1-specific V H Hs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device-induced thrombosis., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Patients with liver diseases are in a hypercoagulable state, as evidenced by enhanced in vitro thrombin generating capacity and elevated plasma levels of markers of in vivo thrombin generation. However, it is unknown by which mechanism in vivo activation of coagulation occurs., Objectives: We aimed to clarify the mechanisms underlying enhanced in vivo thrombin generation to provide a rationale for targeted anticoagulant therapy., Patients/methods: Overall, 191 patients diagnosed with stable or acutely decompensated cirrhosis, acute liver failure or injury, acute-on-chronic liver failure, or sepsis without underlying chronic liver disease were recruited from King's College Hospital, London, from 2017 to 2021 and compared with reference values of 41 healthy controls. We measured levels of markers of in vivo activation of coagulation and activation of the intrinsic and extrinsic pathways, their respective zymogens, and natural anticoagulants., Results: Thrombin-antithrombin complexes, prothrombin fragment 1+2 (F1+2), and D-dimer levels were increased in acute and chronic liver disease, proportional to disease severity. Plasma levels of free activated factor XII (FXIIa), C1-esterase-inhibitor (C1inh)-FXIIa, C1inh-factor XI, C1inh-plasma kallikrein, factor-VIIa-antithrombin-complexes, and activated FVII were reduced in acute and chronic liver disease, even after adjusting for zymogen levels, which were also substantially reduced. Natural anticoagulants antithrombin and protein C were profoundly reduced in liver patients., Conclusions: This study provides evidence of enhanced thrombin generation in liver disease without detectable activation of the intrinsic or extrinsic pathway. We propose that defective anticoagulant mechanisms highly amplify the low-grade activation of coagulation by either pathway., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recombinant human tissue plasminogen activator (rh-tPA) is an important thrombolytic agent for treatment of acute ischemic stroke. It requires fibrin binding for plasminogen activation. In contrast, Microlyse, a novel thrombolytic agent, requires von Willebrand factor (VWF) binding for plasminogen activation. We compared rh-tPA with Microlyse, administered 20 minutes after inducing thrombosis, in 2 randomized blinded acute ischemic stroke mouse models. Thrombosis was induced in the middle cerebral artery with different experimental triggers. Where thrombin infusion generates fibrin-rich thrombi, topical FeCl3 application generates platelet-rich thrombi. In the fibrin-rich model, both rh-tPA and Microlyse increased cortical reperfusion (determined by laser speckle imaging) 10 minutes after therapy administration (35.8 ± 17.1%; P = .001 39.3 ± 13.1%; P < .0001; 15.6 ± 7.5%, respectively, vs vehicle). In addition, both thrombolytic agents reduced cerebral lesion volume (determined by magnetic resonance imaging) after 24 hours (18.9 ± 11.2 mm3; P = .033; 16.1 ± 13.9 mm3; P = .018; 26.6 ± 5.6 mm3, respectively, vs vehicle). In the platelet-rich model, neither rh-tPA nor Microlyse increased cortical reperfusion 10 minutes after therapy (7.6 ± 8.8%; P = .216; 16.3 ± 13.9%; P = .151; 10.1 ± 7.9%, respectively, vs vehicle). However, Microlyse, but not rh-tPA, decreased cerebral lesion volumes (13.9 ± 11.4 mm3; P < .001; 23.6 ± 11.1 mm3; P = .188; 30.3 ± 10.9 mm3, respectively, vs vehicle). These findings support broad applicability of Microlyse in ischemic stroke, irrespective of the thrombus composition., (© 2022 by The American Society of Hematology.)

Background: Thrombosis is a frequent and severe complication in patients with coronavirus disease 2019 (COVID-19) admitted to the intensive care unit (ICU). Lupus anticoagulant (LA) is a strong acquired risk factor for thrombosis in various diseases and is frequently observed in patients with COVID-19. Whether LA is associated with thrombosis in patients with severe COVID-19 is currently unclear., Objective: To investigate if LA is associated with thrombosis in critically ill patients with COVID-19., Patients/methods: The presence of LA and other antiphospholipid antibodies was assessed in patients with COVID-19 admitted to the ICU. LA was determined with dilute Russell's viper venom time (dRVVT) and LA-sensitive activated partial thromboplastin time (aPTT) reagents., Results: Of 169 patients with COVID-19, 116 (69%) tested positive for at least one antiphospholipid antibody upon admission to the ICU. Forty (24%) patients tested positive for LA; of whom 29 (17%) tested positive with a dRVVT, 19 (11%) tested positive with an LA-sensitive aPTT, and 8 (5%) tested positive on both tests. Fifty-eight (34%) patients developed thrombosis after ICU admission. The odds ratio (OR) for thrombosis in patients with LA based on a dRVVT was 2.5 (95% confidence interval [CI], 1.1-5.7), which increased to 4.5 (95% CI, 1.4-14.3) in patients at or below the median age in this study (64 years). LA positivity based on a dRVVT or LA-sensitive aPTT was only associated with thrombosis in patients aged less than 65 years (OR, 3.8; 95% CI, 1.3-11.4) and disappeared after adjustment for C-reactive protein., Conclusion: Lupus anticoagulant on admission is strongly associated with thrombosis in critically ill patients with COVID-19, especially in patients aged less than 65 years., (© 2022 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Background: Severe acute respiratory syndrome coronavirus 2 infection is associated with an increased incidence of thrombosis., Objectives: By studying the fibrin network structure of coronavirus disease 2019 (COVID-19) patients, we aimed to unravel pathophysiological mechanisms that contribute to this increased risk of thrombosis. This may contribute to optimal prevention and treatment of COVID-19 related thrombosis., Patients/methods: In this case-control study, we collected plasma samples from intensive care unit (ICU) patients with COVID-19, with and without confirmed thrombosis, between April and December 2020. Additionally, we collected plasma from COVID-19 patients admitted to general wards without thrombosis, from ICU patients with pneumococcal infection, and from healthy controls. Fibrin fiber diameters and fibrin network density were quantified in plasma clots imaged with stimulated emission depletion microscopy and confocal microscopy. Finally, we determined the sensitivity to fibrinolysis., Results: COVID-19 ICU patients (n = 37) and ICU patients with pneumococcal disease (n = 7) showed significantly higher fibrin densities and longer plasma clot lysis times than healthy controls (n = 7). No differences were observed between COVID-19 ICU patients with and without thrombosis, or ICU patients with pneumococcal infection. At a second time point, after diagnosis of thrombosis or at a similar time point in patients without thrombosis, we observed thicker fibers and longer lysis times in COVID-19 ICU patients with thrombosis (n = 19) than in COVID-19 ICU patients without thrombosis (n = 18)., Conclusions: Our results suggest that severe COVID-19 is associated with a changed fibrin network structure and decreased susceptibility to fibrinolysis. Because these changes were not exclusive to COVID-19 patients, they may not explain the increased thrombosis risk., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Thrombomodulin on endothelial cells can form a complex with thrombin. This complex has both anticoagulant properties, by activating protein C, and clot-protective properties, by activating thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI (TAFIa) inhibits plasmin-mediated fibrinolysis., Objectives: TAFIa inhibition is considered a potential antithrombotic strategy. So far, this goal has been pursued by developing compounds that directly inhibit TAFIa. In contrast, we here describe variable domain of heavy-chain-only antibody (VhH) clone 1 that inhibits TAFI activation by targeting human thrombomodulin., Methods: Two llamas (Lama Glama) were immunized, and phage display was used to select VhH anti-thrombomodulin (TM) clone 1. Affinity was determined with surface plasmon resonance and binding to native TM was confirmed with flow cytometry. Clone 1 was functionally assessed by competition, clot lysis, and thrombin generation assays. Last, the effect of clone 1 on tPA-mediated fibrinolysis in human whole blood was investigated in a microfluidic fibrinolysis model., Results: VhH anti-TM clone 1 bound recombinant TM with a binding affinity of 1.7 ± 0.4 nM and showed binding to native TM. Clone 1 competed with thrombin for binding to TM and attenuated TAFI activation in clot lysis assays and protein C activation in thrombin generation experiments. In a microfluidic fibrinolysis model, inhibition of TM with clone 1 fully prevented TAFI activation., Discussion: We have developed VhH anti-TM clone 1, which inhibits TAFI activation and enhances tPA-mediated fibrinolysis under flow. Different from agents that directly target TAFIa, our strategy should preserve direct TAFI activation via thrombin., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Pulmonary embolism (PE) occurs in one-third of critically-ill COVID-19 patients. Although prior studies identified several pathways contributing to thrombogenicity, it is unknown whether this is COVID-19-specific or also occurs in ARDS patients with another infection., Objective: To compare pathway activity among patients having COVID-19 with PE (C19PE+), COVID-19 without PE (C19PE-), and influenza-associated ARDS (IAA) using a targeted proteomics approach., Methods: We exploited an existing biorepository containing daily plasma samples to carefully match C19PE+ cases to C19PE- and IAA controls on mechanical ventilation duration, PEEP, FiO2, and cardiovascular-SOFA (n = 15 per group). Biomarkers representing various thrombosis pathways were measured using proximity extension- and ELISA-assays. Summed z-scores of individual biomarkers were used to represent total pathway activity., Results: We observed no relevant between-group differences among 22 biomarkers associated with activation of endothelium, platelets, complement, coagulation, fibrinolysis or inflammation, except sIL-1RT2 and sST2, which were lower in C19PE- than IAA (log2-Foldchange -0.67, p = .022 and -1.78, p = .022, respectively). However, total pathway analysis indicated increased activation of endothelium (z-score 0.2 [-0.3-1.03] vs. 0.98 [-2.5--0.3], p = .027), platelets (1.0 [-1.3-3.0] vs. -3.3 [-4.1--0.6], p = .023) and coagulation (0.8 [-0.5-2.0] vs. -1.0 [-1.6-1.0], p = .023) in COVID-19 patients (C19PE+/C19PE- groups combined) compared to IAA., Conclusion: We observed only minor differences between matched C19PE+, C19PE-, and IAA patients, which suggests individual biomarkers mostly reflect disease severity. However, analysis of total pathway activity suggested upregulation of some distinct processes in COVID-19 could be etiologically related to increased PE-risk., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Occlusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN-TaSER), consisting of a function-blocking V H H against glycoprotein Ibα (GPIbα) and a thrombin-inhibiting serine protease inhibitor (SERPIN; α1-antitrypsin 355 AIAR 358 ) to interfere with platelet-driven thrombin formation., Aim: To evaluate the antithrombotic properties of TaSER., Methods: Besides TaSER, we generated three analogous control variants with either a wild-type antitrypsin subunit, a non-targeting control V H H, or their combination. We investigated TaSER and controls in protease activity assays, (platelet-dependent) thrombin generation assays, and by western blotting. The effects of TaSER on platelet activation and von Willebrand factor (VWF) binding were studied by fluorescence-activated cell sorting, in agglutination studies, and in ATP secretion experiments. We studied the influence of TaSER in whole blood (1) on platelet adhesion on VWF, (2) aggregate formation on collagen, and (3) thrombus formation (after recalcification) on collagen and tissue factor., Results: TaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor-triggered thrombin generation and ATP secretion in platelet-rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet-dependent thrombin activity., Conclusion: The synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease., (© 2022 by The American Society of Hematology.)

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis.

The incidence of venous thrombosis, mostly pulmonary embolism (PE), ranging from local immunothrombosis to central emboli, but also deep vein thrombosis (DVT) in people with coronavirus disease 2019 (COVID-19) is reported to be remarkably high. The relevance of better understanding, predicting, treating, and preventing COVID-19-associated venous thrombosis meets broad support, as can be concluded from the high number of research, review, and guideline papers that have been published on this topic. The Dutch COVID & Thrombosis Coalition (DCTC) is a multidisciplinary team involving a large number of Dutch experts in the broad area of venous thrombosis and hemostasis research, combined with experts on virology, critically ill patients, pulmonary diseases, and community medicine, across all university hospitals and many community hospitals in the Netherlands. Within the consortium, clinical data of at least 5000 admitted COVID-19-infected individuals are available, including substantial collections of biobanked materials in an estimated 3000 people. In addition to considerable experience in preclinical and clinical thrombosis research, the consortium embeds virology-hemostasis research models within unique biosafety facilities to address fundamental questions on the interaction of virus with epithelial and vascular cells, in relation to the coagulation and inflammatory system. The DCTC has initiated a comprehensive research program to answer many of the current questions on the pathophysiology and best anticoagulant treatment of COVID-19-associated thrombotic complications. The research program was funded by grants of the Netherlands Thrombosis Foundation and the Netherlands Organization for Health Research and Development. Here, we summarize the design and main aims of the research program., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

The contact system produces the inflammatory peptide bradykinin and contributes to experimental thrombosis. C1 esterase-inhibitor (C1INH) deficiency or gain-of-function mutations in factor XII (FXII) cause hereditary angioedema, a life-threatening tissue swelling disease. C1INH is a relatively weak contact system enzyme inhibitor. Although α1-antitrypsin (α1AT) does not naturally inhibit contact system enzymes, a human mutation (M358R; α1AT-Pittsburgh) changes it into a powerful broad-spectrum enzyme inhibitor. It blocks the contact system, but also thrombin and activated protein C (APC), making it an unattractive candidate for therapeutic contact system blockade. We adapted the reactive center loop of α1AT-Pittsburgh (AIPR/S) to overcome these obstacles. Two α1AT variants (SMTR/S and SLLR/S) strongly inhibit plasma kallikrein, activated FXII, and plasmin. α1AT-SMTR/S no longer inhibits thrombin, but residually inhibits APC. In contrast, α1AT-SLLR/S residually inhibits thrombin, but no longer APC. Additional modification at the P1' position (S→V) eliminates residual inhibition of thrombin and APC for both variants, while retaining their properties as contact system inhibitors. Both α1AT-SMTR/V and -SLLR/V are superior to C1INH in reducing bradykinin production in plasma. Owing to their capacity to selectively block contact system-driven coagulation, both variants block vascular occlusion in an in vivo model for arterial thrombosis. Furthermore, both variants block acute carrageenan-induced tissue edema in mice. Finally, α1AT-SLLR/V, our most powerful candidate, suppresses epithelial leakage of the gut in a mouse model of colitis. Our findings confirm that redesign of α1AT strongly alters its inhibitory behavior and can be used for the treatment of contact system-mediated thrombosis and inflammation., (© 2019 by The American Society of Hematology.)

Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. The factor XII-driven contact system starts coagulation and inflammatory mechanisms via the intrinsic pathway of coagulation and the bradykinin-producing kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. Challenging the concept of the coagulation balance, targeting factor XII or its activator polyphosphate, provides protection from thromboembolic diseases without interfering with hemostasis. This suggests that the polyphosphate/factor XII axis contributes to thrombus formation while being dispensable for hemostatic processes. In contrast to deficiency in factor XII providing safe thromboprotection, excessive FXII activity is associated with the life-threatening inflammatory disorder hereditary angioedema. The current review summarizes recent findings of the polyphosphate/factor XII-driven contact system at the intersection of procoagulant and proinflammatory disease states. Elucidating the contact system offers the exciting opportunity to develop strategies for safe interference with both thrombotic and inflammatory disorders., (© 2018 by The American Society of Hematology.)

This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advances, and basic clotting factor biology. Included in this review article are illustrated abstracts provided by our speakers, which highlight the main conclusions of each invited talk. This will be the first meeting without Dr. Roberts in attendance, yet his commitment to excellent science and his focus on turning science to patient care are pervasively reflected in the presentations by our speakers.

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII., (© 2017 by The American Society of Hematology.)

In epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling.

Coagulation factor XII (FXII, Hageman factor, EC = 3.4.21.38) is the zymogen of the serine protease, factor XIIa (FXIIa). FXII is converted to FXIIa through autoactivation induced by "contact" to charged surfaces. FXIIa is of crucial importance for fibrin formation in vitro, but deficiency in the protease is not associated with excessive bleeding. For decades, FXII was considered to have no function for coagulation in vivo. Our laboratory developed the first murine knockout model of FXII. Consistent with their human counterparts, FXII(-/-) mice have a normal hemostatic capacity. However, thrombus formation in FXII(-/-) mice is largely defective, and the animals are protected from experimental cerebral ischemia and pulmonary embolism. This murine model has created new interest in FXII because it raises the possibility for safe anticoagulation, which targets thrombosis without influence on hemostasis. We recently have identified platelet polyphosphate (an inorganic polymer) and mast cell heparin as in vivo FXII activators with implications on the initiation of thrombosis and edema during hypersensitivity reactions. Independent of its protease activity, FXII exerts mitogenic activity with implications for angiogenesis. The goal of this review is to summarize the in vivo functions of FXII, with special focus to its functions in thrombosis and vascular biology.

The aim of this study was to determine the sensitivity of transgenic immune tolerant mice for the type and level of aggregation of recombinant human interferon alpha2b (rhIFNalpha2b). RhIFNalpha2b was aggregated by metal-catalyzed oxidation or by incubation at elevated temperature and various pHs. Native rhIFNalpha2b was mixed with oxidized rhIFNalpha2b at different ratios to obtain samples with different aggregation levels. The preparations were characterized by UV and fluorescence spectroscopy, gel permeation chromatography (GPC), dynamic light scattering (DLS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and ELISA. The immunogenicity was evaluated in wild-type mice and transgenic mice immune tolerant for hIFNalpha2. Sera were analyzed by ELISA for the presence of rhIFNalpha2b-specific antibodies. The oxidized and aged preparations widely differed regarding the level and nature of aggregates. All preparations containing aggregates increased the immune response in the wild-type mice as compared to native rhIFNalpha2b and were able to break the tolerance of the transgenic mice. The more native-like the conformation of the aggregated proteins, the more immunogenic the preparations were in the transgenic mice. The native-like aggregates prepared via metal catalysis induced a dose-dependent loss of tolerance in the transgenic mice. In conclusion, the transgenic mouse model can be used to screen rhIFNalpha2b formulations for low levels of immunogenic aggregates obtained under accelerated storage conditions., ((c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association)