Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO) or Pdcd10 (Pdcd10ECKO), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.
Background: Activated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3.Objective: To date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C-terminus of the light chain have not been studied in detail. Methods:Site-directed Ala-scanning mutagenesis of six positively charged residues within G142-L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities.Results and Conclusions: K151 was involved in protein S dependent-anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S-dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C-terminal region of APC's light chain overlap. K E Y W O R D S activated protein C, anticoagulant activity, cytoprotective activity, protease activated receptor, protein S S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section.
Primary prophylaxis is a method of haemostatic management to prevent bleeding and arthropathy in patients with severe haemophilia. The aim of this study was to evaluate the usefulness of primary prophylaxis in patients with severe haemophilia A. This study included 15 patients with haemophilia A who received primary prophylaxis at our institution for a minimum of 5 years. We evaluated the annualized bleeding ratio of joints or other sites, current joint function, and X-ray images and MRI scans taken when patients were 6 years old. The range of patients' ages at the end of the study was 6.2-16.8 years, and at the start of primary prophylaxis it was 0.8-2.4 years. Factor VIII concentrates (25-40 units kg(-1) dose(-1)) were administered 3 times/week or every other day, according to the Swedish protocol. Mean joint and non-joint annualized bleeding ratios were 0.49 ± 0.5 and 1.54 ± 1.69, respectively. At the final evaluation, all patients displayed a normal range of motion for both elbows, knees, and ankles. The radiography and MRI findings at the age of 6 were unremarkable in all patients. Overall, primary prophylaxis for patients with severe haemophilia A was performed safely, reduced the number of bleeding events, and prevented progression to arthropathy.
Background: Increased coagulation factor VIII (FVIII:C) activity is potentially associated with the pathogenesis of glucocorticoid (GC)-induced coagulopathy. However, the mechanism underlying the increase in FVIII:C activity remains unclear. Objectives: We analyzed the changes in coagulation parameters, including FVIII and von Willebrand factor (VWF), in pediatric patients with immune thrombocytopenic purpura (ITP) treated with GC. The influence of GC treatment on mRNA expression of the FVIII (F8) and VWF (VWF) genes was examined in human liver sinusoidal endothelial cells (HLSEC/ciJ) and umbilical vein endothelial cells (EA. hy926). Methods: Activated partial thromboplastin time (APTT) and levels of FVIII:C, FVIII antigen (FVIII:Ag), and VWF antigen (VWF:Ag) were compared before and after GC treatment in patients with ITP. Changes in F8 and VWF mRNA levels in HLSEC/ciJ and EA. hy926 cells before and after dexamethasone (DEX) treatment were quantified by reverse transcription polymerase chain reaction. Changes in FVIII protein levels were also evaluated in the lysates of DEX-treated HLSEC/ciJ cells. Results: Values of APTT significantly decreased after GC treatment in ITP patients; significant increases in FVIII:C and FVIII:Ag levels were observed (p<0.05). Notably, no significant increase in VWF:Ag level was observed. F8 and VWF mRNA expressions in HLSEC/ciJ cells increased following DEX treatment (1 and 100 μM) (p<0.05), with the most significant increase seen in F8 mRNA levels (100 μM DEX; p<0.01). These increases were nullified by pretreatment with the GC receptor (GR) antagonist RU486. Additionally, VWF mRNA, but not F8 mRNA, expression increased after treating EA. hy926 cells with 1 μM DEX (p<0.05). No significant increase in FVIII protein level was observed in HLSEC/ciJ cells after treatment with 100 μM DEX. Conclusions: Direct enhancement of mRNA levels of endogenous FVIII by GC via GR activation is, at least in part, a mechanism underlying the increase in FVIII levels during GC treatment.
Hemophilia B Leyden is a unique subtype of hemophilia B, characterized by increasing factor IX activity (FIX:C) after puberty and a lower normal range of FIX:C throughout adulthood. However, to date, no Japanese case has been reported. Here, we report a case of hemophilia B Leyden in a 22-year-old male. He suffered from subgaleal hematoma, and was subsequently diagnosed with hemophilia B (FIX:C 0.2%) in the neonatal period. Both his parents are Japanese. There was no history of hemophilia in his family. FIX:C gradually increased with age (8% at age = 1; 14% at age = 7; 19% at age = 12; 32% at age = 18). FIX:C is within the range 30-40% in recent several years. He once required administration of FIX concentrate against traumatic tongue bleeding at 7 years of age. Genotyping analysis of FIX was performed after informed consent at 21 years of age, and a point mutation (c.-35G>A) was detected. This mutation has been reported previously as the Leyden mutation. Although it has been reported that hemophilia B Leyden is seen in 1.9% of patients with hemophilia B, the present case is the first report of hemophilia B Leyden from Japan.
Unfractionated heparin (UFH) is widely used in the treatment and prophylaxis of thrombosis. The anticoagulant effect of UFH is monitored according to activated partial thromboplastin time (APTT). However, there are reports of cases in which hemorrhagic complications develop despite APTT being maintained within the therapeutic range. This indicates there are problems with the monitoring method using APTT during UFH treatment. To assess the actual anticoagulant effect in UFH therapy using APTT as a monitoring method, we investigated changes in APTT and thrombin generation (TG) potential in vitro with the addition of UFH to samples from 10 healthy adults. There were large individual differences in the degree of APTT prolongation with the addition of UFH to samples from healthy adults. Furthermore, individual differences in the degree of TG change exceeded those in APTT in 7 subjects with comparable APTT changes caused by UFH (coefficients of variation: 2-5% for APTT, 10-50% for TG potential). Thus, with UFH treatment, there are large individual differences in the degree of TG inhibition even when APTT is within the appropriate range for coagulation control. These findings provide invaluable information for resolving the problem of hemorrhagic complications during UFH treatment based on the current APTT method.
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