Novel cysteine substitution p.(Cys1084Tyr) causes variable expressivity of qualitative and quantitative VWF defects

Von Willebrand factor (VWF) is an extremely cysteine-rich multimeric protein that is essential for maintaining normal hemostasis. The cysteine residues of VWF monomers form intra- and inter-molecular disulfide bonds that regulate its structural conformation, multimer distribution and ultimately its hemostatic activity. In this study we investigated and characterized the molecular and pathogenic mechanisms through which a novel cysteine variant p.(Cys1084Tyr) causes an unusual, mixed phenotype form of von Willebrand disease (VWD). Phenotypic data including bleeding scores, laboratory values, VWF multimer distribution and desmopressin response kinetics were investigated in 5 members (2 parents and 3 daughters) of a consanguineous family. VWF synthesis and secretion were also assessed in a heterologous expression system and in a transient transgenic mouse model. Heterozygosity for p.(Cys1084Tyr) was associated with variable expressivity of qualitative VWF defects. Heterozygous individuals had reduced VWF:GPIbM (<0.40IU/mL) and VWF:CB (<0.35IU/mL) as well as relative reductions in high-molecular weight multimers, consistent with type 2A VWD. In addition to these qualitative defects, homozygous individuals also displayed reduced FVIII:C/VWF:Ag leading to very low FVIII levels (0.03-0.1IU/mL) as well as reduced VWF:Ag (<0.40IU/mL) and VWF:GPIbM (<0.30IU/ml). Accelerated VWF clearance and impaired VWF secretion contributed to the fully expressed homozygous phenotype with impaired secretion arising due to disordered disulfide connectivity.

Functional Characteristics and Regulated Expression of Alternatively Spliced Tissue Factor: An Update

In human and mouse, alternative splicing of tissue factor’s primary transcript yields two mRNA species: one features all six TF exons and encodes full-length tissue factor (flTF), and the other lacks exon 5 and encodes alternatively spliced tissue factor (asTF). flTF, which is oftentimes referred to as “TF”, is an integral membrane glycoprotein due to the presence of an alpha-helical domain in its C-terminus, while asTF is soluble due to the frameshift resulting from the joining of exon 4 directly to exon 6. In this review, we focus on asTF—the more recently discovered isoform of TF that appears to significantly contribute to the pathobiology of several solid malignancies. There is currently a consensus in the field that asTF, while dispensable to normal hemostasis, can activate a subset of integrins on benign and malignant cells and promote outside-in signaling eliciting angiogenesis; cancer cell proliferation, migration, and invasion; and monocyte recruitment. We provide a general overview of the pioneering, as well as more recent, asTF research; discuss the current concepts of how asTF contributes to cancer progression; and open a conversation about the emerging utility of asTF as a biomarker and a therapeutic target.

Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets

Vincristine is a clinically used antimicrotubule drug for treating patients with lymphoma. Due to its property of increasing platelet counts, vincristine is also used to treat patients with immune thrombocytopenia. Moreover, antiplatelet agents were reported to be beneficial in thrombotic thrombocytopenic purpura (TTP). Therefore, we investigated the detailed mechanisms underlying the antiplatelet effect of vincristine. Our results revealed that vincristine inhibited platelet aggregation induced by collagen, but not by thrombin, arachidonic acid, and the thromboxane A2 analog U46619, suggesting that vincristine exerts higher inhibitory effects on collagen-mediated platelet aggregation. Vincristine also reduced collagen-mediated platelet granule release and calcium mobilization. In addition, vincristine inhibited glycoprotein VI (GPVI) signaling, including Syk, phospholipase Cγ2, protein kinase C, Akt, and mitogen-activated protein kinases. In addition, the in vitro PFA-100 assay revealed that vincristine did not prolong the closure time, and the in vivo study tail bleeding assay showed that vincristine did not prolong the tail bleeding time; both findings suggested that vincristine may not affect normal hemostasis. In conclusion, we demonstrated that vincristine exerts antiplatelet effects at least in part through the suppression of GPVI signaling. Moreover, this property of antiplatelet activity of vincristine may provide additional benefits in the treatment of TTP.

The Biological Significance of von Willebrand Factor O-Linked Glycosylation

Glycosylation is a key posttranslational modification, known to occur on more than half of all secreted proteins in man. As such, the role of N- and O-linked glycan structures in modulating various aspects of protein biology is an area of much research. Given their prevalence, it is perhaps unsurprising that variations in glycan structures have been demonstrated to play critical roles in modulating protein function and have been implicated in the pathophysiology of human diseases. von Willebrand factor (VWF), a plasma glycoprotein that is essential for normal hemostasis, is heavily glycosylated, containing 13 N-linked and 10 O-linked glycans. Together, these carbohydrate chains account for 20% of VWF monomeric mass, and have been shown to modulate VWF structure, function, and half-life. In this review, we focus on the specific role played by O-linked glycans in modulating VWF biology. Specifically, VWF O-linked glycans have been shown to modulate tertiary protein structure, susceptibility to ADAMTS13 proteolysis, platelet tethering, and VWF circulatory half-life.

Management Dilemmas in Patients with Traumatic Brain Injury on Anticoagulants

Introduction A normal individual with normal hemostasis maintains a balance between thrombus formation and destruction using a complex interaction between the smooth vascular endothelium, the coagulation cascade, the platelet aggregation system, and the fibrinolysis mechanism. However, in patients who are on either antiplatelet drugs (APDs) or anticoagulants (ACDs), this normal homeostasis is altered. This is further altered with traumatic brain injury (TBI) and thus, we need specific guidelines to address this subpopulation to decide the length of observation, avoid unnecessary hospitalization, and relieve the economic burden. There exists a very few randomized controlled trials (RCTs) for this clinical question and a thorough risk–benefit analysis for each patient is prudent before making clinical decisions. Materials and Methods This is a review article based on available evidence published in literature. Results There are multiple therapeutic drugs which act on various stages of the coagulation mechanism. These include antiplatelet agents, Vitamin K antagonists, Heparin, Antithrombin III, and Glycoprotein IIb/IIIA inhibitors. While the initial management of head injuries depends on the severity of head injuries, management of head injuries in patients on anticoagulants needs to be approached with care and caution. There are multiple dilemmas including role of CT scans, duration and reason for admission, when to restart anticoagulation, etc. We suggest the recommendations based on available literature; however, no evidence can be given as these are not based on any RCTs, due to paucity of such studies. Conclusion The guidelines are based on previously conducted trials and consensus. We have attempted to provide a pragmatic and practical approach to such cases with the hope that it will ensure minimum risks with the best possible patient outcomes. The entire journey from patient presentation to follow-up has been covered in this article and we hope this would be useful to all practicing in the field of neurotrauma.

Blood coagulation in the 21st century: existing knowledge, current strategies for treatment and perspective

Disorders in the blood coagulation system are the leading cause of death and disability in the modern world. So the search for new drugs that can prevent pathological thrombosis, while not affecting normal hemostasis, becomes more relevant than ever. Recent studies has been a revolution in the understanding of the principles of work and the regulation of blood coagulation. In addition, new, more effective approaches to drug development have now appeared. For example computer simulation methods that can significantly reduce the time and resources spent on the search for new candidate molecules. In the review, the blood clotting system, the molekular mechanisms of thrombosis, the role of blood coagulation factors Xa and XIa, and the urgency of developing new inhibitors of these targets are shown, and the most interesting inhibitors of factors Xa and XIa are presented.

Coagulopathy in the Acute Phase of Traumatic Brain Injury

Acute traumatic brain injury (TBI) can be accompanied by coagulopathy. In TBI, when an increased risk of hemorrhagic lesions in the brain tissue exists, keeping the normal hemostasis is crucial.The aim: to determine the incidence of coagulopathy in the acute phase of isolated TBI and identify the correlation between the types of hemostasis disorders and the severity of TBI.Materials and methods. We analyzed 323 case records of patients with TBI hospitalized to the Neurosurgery Center ICU from 2008 to 2016, 118 of them were included in the study. Criteria for inclusion were acute isolated TBI, hospitalization in the first 72 hours after injury, young and middle-aged patients. On admission to the hospital, all patients were assessed according to the Glasgow Coma Scale (GCS) and divided into two groups: group I with severe TBI, group 2 with mild and moderate TBI. All patients underwent blood coagulation testing (APTT, PTI, fibrinogen concentration, platelet count).Results. In 63.6% of patients with acute TBI coagulopathy were found, most of them (55.1%) had hypocoagulability state and were observed in the group of severe PMT (62.5%). The most frequent signs of coagulopathy were reduced PTI of less than 70% and thrombocytopenia of less than 150×109/l. A weak correlation (R=0.276; P=0.002) was found between the development of thrombocytopenia and adverse outcomes (1–3 points according to GOS).Conclusion. on hospital admission coagulopathy was diagnosed in 63,6% of patients in the acute phase of TBI, hypocoagulability state prevailed. Coagulopathy was significantly more common in patients in the acute phase of severe TBI (GCS8 points).The mechanisms of TBI-associated coagulopathy have not yet been fully clarified. Further studies of the role of platelet, plasma and fibrinolytic components in the development of coagulopathy are warranted, which require more comprehensive methods of hemostasis investigation.

The protein C activator AB002 rapidly interrupts thrombus development in baboons

Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin’s procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060.

VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

NADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.