Rare bleeding disorders-old diseases in the era of novel options for therapy.

Rare diseases are defined as life-threatening or chronically debilitating diseases with a prevalence of less than one per 2000 according to the European Union or one per 1250 according to the USA. Congenital rare bleeding disorders RBD are reported in most populations, with incidence varying from 1 in 5000 (Hemophilia A), 1:30,000 (Hemophilia B) to much rarer (1:500,000 for FVII deficiency, 1-3 million for Prothrombin or FXIII deficiency). Acquired Hemophilia A is also a rare bleeding disorder with estimated frequency of 1 in million. Most RBDs are inherited as autosomal recessive (AR); however, heterozygous carriers with varying degrees of corresponding factor deficiency may render an unpredictable propensity for bleeding. In patients with bleeding symptoms, laboratory assessment and especially molecular techniques currently enable accurate diagnosis and may provide tools for prenatal and family counseling. Currently hemostasis control is mainly based upon replacement of the missing coagulation factors (unless presence of inhibitors renders it impossible), however future gene therapy and disruptive, non-replacement alternatives may be promising for patients with RBD.

Applying a direct aPTT ratio (PlatelinLS/ActinFS) permits to identify rapidly and reliably a bleeding-related factor deficiency or a lupus anticoagulant sequential to an isolated prolongation of aPTT in paediatric pre-operative screening.

OBJECTIVES: An isolated prolongation of activated partial thromboplastin time (aPTT) found in paediatric pre-operative screening could be due to bleeding or non-bleeding aetiologies. The aim of our study was to evaluate clinical benefits of an additional ActinFS and/or a mixing aPTT study to identify a bleeding-related factor deficiency (BRFD). METHODS: Over a 4-yr period, isolated prolongation of aPTT with PlatelinLS was detected in 308 paediatric patients and confirmed in 161 cases by a 2nd sample. ActinFS, a mixing study, FVIII, FIX, FXI, FXII and lupus anticoagulant (LA) were performed. Three different aPTT ratios between PlatelinLS and ActinFS were analysed. RESULTS: We found 17 BRFD, 31 FXII deficiencies and 64 positive LA. A prolonged ActinFS had a significant association with BRFD (P < 0.0001) while a corrected mixing study did not. The direct aPTT ratio had a significant relationship with positive LA (P < 0.05), and with BRFD (P < 0.0001). Using this ratio, the sensitivity of BRFD's and LA's detection could be increased, respectively, to 82% from 59% using ActinFS alone, and to 86% from 55% using mixing study. CONCLUSIONS: Applying this direct aPTT ratio allows to quickly and reliably identify both BRFD and LA sequential to an isolated prolongation of aPTT.

[Pseudoxanthoma elasticum-like disease with deficiency of vitamin K-dependent clotting factors and cutis laxa features]. / Pseudoxanthome élastique variant avec déficit en facteurs de coagulation vitamine K-dépendants et perte d'élasticité cutanée.

BACKGROUND: Pseudoxanthoma elasticum (PXE)-like syndrome is characterized by the association of PXE and cutis laxa (CL) features with a deficiency of vitamin K-dependent clotting factors. It was first described in 1971 and was identified as a distinct genetic entity in 2007 with analysis of the GGCX (γ-glutamyl carboxylase) gene, which is involved in congenital deficiency in vitamin K-dependent clotting factors. Here we report a new case of this extremely rare syndrome. PATIENTS AND METHODS: A 23-year-old female patient was seen for the emergence of loose and redundant skin following extensive weight loss. She also presented a deficiency of vitamin K-dependent clotting factors. Physical examination revealed excessive, leathery skin folds in the axillary and neck regions. A skin biopsy revealed polymorphous and fragmented elastic fibers in the reticular dermis. These were mineralized, as was demonstrated by Von Kossa staining. The clinical features of CL associated with the histopathological features of PXE and vitamin K-dependent clotting factor deficiency led us to a diagnosis of PXE-like syndrome. A molecular study of the GGCX gene showed compound heterozygosity. DISCUSSION: The GGCX gene is usually responsible for PXE-like syndrome. GGCX encodes a γ-glutamyl carboxylase necessary for activation of gla-proteins. Gla-proteins are involved both in coagulation factors in the liver and in the prevention of ectopic mineralization of soft tissues. Uncarboxylated forms of gla-proteins in fibroblast would thus enable mineralization and fragmentation of elastic fibers.

[The diagnostic value of protein induced by vitamin K absence or antagonist-II in non-infant patients with acquired deficiency of vitamin K-dependent coagulation factors].

OBJECTIVE: To explore the diagnostic value of protein induced by vitamin K absence or antagonist -II(PIVKA-II) in non-infant with acquired deficiency of vitamin K-dependent coagulation factors(ADVKCF). METHODS: PIVKA-II levels were measured by ELISA in 50 patients with ADVKCF on day 0, 3, 7 after vitamin K treatment. Prothrombin time(PT), APTT, FII: C, FVII: C, FIX: C, and FX: C were analyzed simultaneously. Twenty healthy subjects were enrolled as controls. RESULTS: The average level of PIVKA-II in ADVKCF group was (3.83 ± 1.40)µg/L, while (1.30 ± 0.54) µg/L in the control group (P < 0.05). The PIVKA-II levels on day 0 and 3 did not show significant difference [(3.83 ± 1.40) µg/L vs (3.79 ± 0.66) µg/L, P > 0.05], but decreasing significantly on day 7 compared to the control group(P < 0.05). The PIVKA-II level was (3.78 ± 1.30) µg/L in patients receiving plasma transfusion, while (3.91 ± 1.49)µg/L in no-plasma-transfusion group (P > 0.05). Coagulation factors II, VII, IX and X activity which decreased significantly before treatment returned to normal range after one week use of vitamin K, leading to complete correction of prolonged APTT and PT (>100 seconds). CONCLUSIONS: The PIVKA-II level in ADVKCF patients is significantly higher than that of healthy subjects within one week treatment of vitamin K, which is not influenced by plasma transfusion. This study suggests that PIVKA-II is a more sensitive parameter than APTT, PT and the activity of coagulation factor, which could be a valuable factor in the early diagnosis of ADVKCF.

[Thrombin generation assays and their clinical application]. / Trombingenerációs vizsgálatok és klinikai alkalmazásuk.

Thrombin is a key enzyme of the coagulation system, having both pro- and anticoagulant functions. Thus, the generation of thrombin is one of the most important steps in coagulation. Global haemostasis assay, the so-called thrombin generation test is appropriate for its assessment. Since thrombin generation is sensible for both pro- and anticoagulant processes it can be applied for the general characterisation of the risk of thrombosis and bleeding, too. Clinical studies confirmed augmented thrombin generation in patients with high risk of venous or arterial thrombosis. Anticoagulant therapy (also novel oral anticoagulant treatment) can be monitored by thrombin generation. In case of haemophilia thrombin generation assays reflect bleeding severity. It is applicable for monitoring of both conventional haemophilia treatment and inhibitor-bypassing therapy, which is needed when inhibitors develop in patients. Standardization of thrombin generation methods and determination of cut off values are required before its application in clinical practice.

Comparative analysis of thrombin generation platforms for patients with coagulation factor deficiencies: A comprehensive assessment.

INTRODUCTION: Thrombin generation assays (TGAs) assess the overall functionality of the hemostatic system and thereby provide a reflection of the hemostatic capacity of patients with disorders in this system. Currently, four (semi-)automated TGA platforms are available: the Calibrated Automated Thrombogram, Nijmegen Hemostasis Assay, ST Genesia and Ceveron s100. In this study, we compared their performance for detecting patients with congenital single coagulation factor deficiencies. MATERIALS AND METHODS: Pooled patient samples, healthy control samples and normal pooled plasma were tested on all four platforms, using the available reagents that vary in tissue factor and phospholipid concentrations. The TGA parameters selected for analysis were peak height and thrombin potential. Results were normalized by using the calculated mean of healthy controls and a correction for between-run variation. Outcomes were presented as relative values, with the mean of healthy controls standardized to 100 %. RESULTS: Across all platforms and reagents used, thrombin potentials and peak heights of samples with coagulation factor deficiencies were lower than those of healthy controls. Reagents designed for bleeding tendencies yielded the lowest values on all platforms (relative median peak height 19-32 %, relative median thrombin potential 19-45 %). Samples representing more severe coagulation factor deficiencies generally exhibited lower relative peak heights and thrombin potentials. CONCLUSIONS: Thrombin generation assays prove effective in differentiating single coagulation factor deficient samples from healthy controls, with modest discrepancies observed between the platforms. Reagents designed for assessing bleeding tendencies, featuring the lowest tissue factor and phospholipid concentrations, emerged as the most suitable option for detecting coagulation factor deficiencies.

Rare bleeding disorders: worldwide efforts for classification, diagnosis, and management.

Rare bleeding disorders (RBDs) comprise the inherited deficiencies of coagulation factors such as fibrinogen, factor (F)II, FV, FV + FVIII, FVII, FX, FXI, and FXIII, and are usually transmitted as autosomal recessive disorders. RBDs are characterized by a wide variety of symptoms from mild to severe; however, due to their rarity, only little information is available on the adequate management of patients affected with these deficiencies. Moreover, the limitations of laboratory assays and the lack of a definitive consensus concerning their classification have prevented adoption of optimal approaches to their individual management. To overcome these limitations, new strategies are therefore necessary, such as the establishment of global collaborations and networks among treatment centers, as well as increasing support provided by public health organizations.

Preoperative plasma hyperfibrinogenemia is predictive of poor prognosis in patients with nonmetastatic colon cancer.

BACKGROUND: The outcomes of colorectal cancer are determined by host factors, including systemic inflammation. The purpose of this study was to evaluate the prognostic significance of fibrinogen and inflammation-based scores, as markers of the inflammatory response, in colon cancer. METHODS: We retrospectively reviewed the medical records of patients with nonmetastatic colon cancer who underwent curative resection between January 2005 and December 2007. Fibrinogen, albumin, C-reactive protein, neutrophil, lymphocyte, and platelet counts were measured at the time of diagnosis. Correlations between preoperative plasma fibrinogen levels and clinicopathologic characteristics were analyzed. Univariate and multivariate survival analyses were performed to identify factors associated with disease-free and overall survival. RESULTS: A total of 624 patients who underwent curative resection for colon cancer were eligible for this study. Mean preoperative plasma fibrinogen levels were 325.24±88.19 mg/dl. Higher preoperative plasma fibrinogen levels were associated with sex (male), old age, poorly/mucinous differentiated tumor, advanced tumor stage, elevated carcinoembryonic antigen (CEA) levels, higher modified Glasgow Prognostic Score, and higher neutrophil:lymphocyte and platelet:lymphocyte ratios. In multivariate analysis, elevated plasma fibrinogen level [disease-free survival: hazard ratio (HR) 1.999, 95% confidence interval (95% CI) 1.081-3.695, P=.027; overall survival: HR 3.138, 95% CI 1.077-9.139, P=.036], advanced tumor stage, and higher CEA levels were independently associated with worse disease-free survival and overall survival. None of the inflammation-based scores were significantly associated with survival. CONCLUSIONS: Fibrinogen as one of inflammatory markers may be considered a possible prognostic marker in colon cancer.

Rare factor deficiencies.

PURPOSE OF REVIEW: By definition, rare factor deficiencies have a prevalence of less than 200,000 in the US population, or an incidence of less than one in 2000 in Europe. The very small numbers of patients with rare disorders present challenges in diagnosis, evaluation of bleeding risk and treatment. Use of new assays, full genome sequencing, and global clotting assays will significantly improve diagnosis of patients with rare bleeding disorders. RECENT FINDINGS: In addition to new assays available for monitoring patients, new therapy, both recombinant and plasma derived, is now available. Registries and clinical trials have demonstrated decreased bleeding and improved outcomes when patients are treated with these agents. Expanding international registries have been initiated to correlate genotype and bleeding phenotype in conjunction with global assays. SUMMARY: Ongoing research continues to expand our understanding of the pathophysiology of rare factor deficiencies. This work complements medical practice to incorporate early diagnosis and new treatment options for patients, resulting in safer and less sensitizing regimens and much improved clinical outcomes.

Clinical and laboratory assessment of the bleeding pediatric patient.

Children presenting with bleeding symptoms pose a diagnostic challenge. The evaluation should include a comprehensive medical and bleeding history, detailed family history, a physical examination, and selected laboratory tests. Evaluation of the bleeding history should include an objective quantitation of the bleeding symptoms, followed by objective quantitation of the family members' bleeding symptoms. If a bleeding disorder is suspected, based on personal and/or family bleeding history, specific laboratory testing is needed for attaining a diagnosis. The screening tests for coagulation factor deficiencies are considered reliable, routinely available, and easy to perform. However, for primary hemostatic defects, the various screening tests have not yet proven to be reliable and thus referral for a specialized hematology consultation and specific laboratory testing are needed. Perhaps a Bayes theorem approach, combining the personal and family bleeding history with the results of a screening test for a primary hemostatic defect would prove to be useful in the identification of those children who will most benefit from specific laboratory testing.

A Clot Twist: Extreme Variation in Coagulotoxicity Mechanisms in Mexican Neotropical Rattlesnake Venoms.

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus, and C. tzabcan. We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus. This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.

Thromboelastography Reaction-Time Thresholds for Optimal Prediction of Coagulation Factor Deficiency in Trauma.

BACKGROUND: Coagulopathy is common in multitrauma patients and repletion of procoagulant factor deficiency with fresh frozen plasma (FFP) improves hemostasis. Optimal kaolin-thromboelastography thresholds for FFP transfusion in trauma patients have not been well established. STUDY DESIGN: Adult trauma patients with an Injury Severity Score ≥15 were included in this retrospective observational cohort study. The primary end point was area under the receiver operating characteristic curve (AUROC) for reaction time (R-time) to detect procoagulant factor deficiency, as reflected by an elevated international normalized ratio (INR) or aPTT. Test characteristics for the optimal R-time threshold calculated in our study were compared against thresholds recommended by the American College of Surgeons for FFP transfusion. RESULTS: Six hundred and ninety-four pairs of thromboelastography and conventional coagulation tests were performed in 550 patients, with 144 patients having additional pairs of tests after the first hour. The R-time was able to detect procoagulant factor deficiency (INR ≥1.5 AUROC 0.80; 95% CI, 0.75 to 0.85; aPTT ≥40 seconds AUROC 0.85; 95% 0.80 to 0.89) and severe procoagulant factor deficiency (INR ≥2.0 AUROC 0.82; 95% CI, 0.73 to 0.99; aPTT ≥60 seconds AUROC 0.89; 95% CI, 0.81 to 0.98) with good accuracy. Optimal thresholds to maximize sensitivity and specificity were 3.9 minutes for detection of INR ≥1.5, 4.1 minutes for detection of aPTT ≥40 seconds, 4.3 minutes for detection of INR ≥2.0, and 4.3 for detection of aPTT ≥60 seconds. Currently recommended R-time thresholds for FFP transfusion had 100% specificity for detecting procoagulant factor deficiency, but low sensitivity (3% to 7%). CONCLUSIONS: R-time can detect procoagulant factor deficiency in multitrauma patients with good accuracy, but currently recommended R-time thresholds are highly specific and not sensitive. Use of low-sensitivity thresholds might result in undertreatment of many patients with procoagulant factor deficiency.

Pharmacological management of rare coagulation factor deficiencies besides hemophilia.

INTRODUCTION: Rare coagulation factor deficiencies are less-known disorders with variable effects on the patient's life. Management of such patients is a challenge due to the paucity of evidence-based data, more so when patients with these rare disorders encounter a more rare, related condition, like inhibitor development or thrombosis. AREA COVERED: A comprehensive literature search related to RCFDs and management was performed in PubMed in order to discuss therapeutic options and challenges, prophylaxis, management of minor and major surgeries, obstetric and gynecological complications, inhibitor development, and thrombosis. EXPERT OPINION: Although significant changes have occurred in the management of RCFDs in recent years, more evidence-based studies besides expert opinion are needed for optimal management.

Two cases of congenital dysfibrinogenemia associated with thrombosis - Fibrinogen Praha III and Fibrinogen Plzen.

Congenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous gamma Y363N mutation (Fibrinogen Praha III) and the heterozygous Aalpha N106D mutation (Fibrinogen Plzen), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzen fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzen. Praha III mutation was situated in the polymerisation pocket "a". The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzen mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector; and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.

Novel splice site mutations in the gamma glutamyl carboxylase gene in a child with congenital combined deficiency of the vitamin K-dependent coagulation factors (VKCFD).

Congenital combined deficiency of the vitamin K-dependent coagulation factors is a rare bleeding disorder caused by either a defect in the gamma-glutamyl carboxylase or the vitamin K epoxide reductase enzyme complex. The diagnosis should be considered when vitamin-K dependent factor activities are decreased and liver dysfunction, vitamin K deficiency, and factitious coumarin ingestion have been excluded. We report a case of VKCFD in a child resulting from compound heterozygosity for two novel splice site mutations of the gamma-glutamyl carboxylase gene. Oral vitamin K supplementation resulted in partial resolution of proteins and complete resolution of bleeding.

Elevated thrombin generation in patients with congenital disorder of glycosylation and combined coagulation factor deficiencies.

BACKGROUND: Congenital disorders of glycosylation are rare inherited diseases affecting many different proteins. The lack of glycosylation notably affects the hemostatic system and leads to deficiencies of both procoagulant and anticoagulant factors. OBJECTIVE: To assess the hemostatic balance in patients with multiple coagulation disorders by using a thrombin generation assay. METHOD: We performed conventional coagulation assays and a thrombin generation assay on samples from patients with congenital disorder of glycosylation. The thrombin generation assay was performed before and after activation of the protein C system by the addition of soluble thrombomodulin. RESULTS: A total of 35 patients were included: 71% and 57% had low antithrombin and factor XI levels, respectively. Protein C and protein S levels were abnormally low in 29% and 26% of the patients, respectively, whereas only 11% displayed low factor IX levels. Under baseline conditions, the thrombin generation assay revealed a significantly higher endogenous thrombin potential and thrombin peak in patients, relative to controls. After spiking with thrombomodulin, we observed impaired involvement of the protein C system. Hence, 54% of patients displayed a hypercoagulant phenotype in vitro. All the patients with a history of stroke-like episodes or thrombosis displayed this hypercoagulant phenotype. CONCLUSION: A thrombin generation assay revealed a hypercoagulant in vitro phenotype under baseline condition; this was accentuated by impaired involvement of the protein C system. This procoagulant phenotype may thus reflect the risk of severe vascular complications. Further research will have to determine whether the thrombin generation assay is predictive of vascular events.

Congenital Factor Deficiencies in Children: A Report of a Single-Center Experience.

Congenital factor deficiencies (CFDs) refer to inherited deficiency of coagulation factors in the blood. A total of 481 patients with CFDs, who were diagnosed and followed at our Pediatric Hematology and Oncology Clinic between 1990 and 2015, were retrospectively evaluated. Of the 481 cases, 134 (27.8%) were hemophilia A, 38 (7.9%) were hemophilia B, 57 (11.8%) were von Willebrand disease (vWD), and 252 (52.3%) were rare bleeding disorders (RBDs). The median age of the patients at the time of diagnosis and at the time of the study was 4.1 years (range: 2 months to 20.4 years) and 13.4 years (range: 7 months to 31.3 years), respectively. The median duration of the follow-up time was 6.8 years (range: 2.5 months to 24.8 years). One hundred nineteen (47.2%) of 252 patients with RBDs were asymptomatic, 49 (41.1%) of whom diagnosed by family histories, 65 (54.6%) through preoperative laboratory studies, and 5 (4.2%) after prolonged bleeding during surgeries. Consanguinity rate for the RBDs was 47.2%. Prophylactic treatment was initiated in 80 patients, 58 of whom were hemophilia A, 7 were hemophilia B, 13 were RBDs, and 2 were vWD. Significant advances have been achieved during the past 2 decades in the treatment of patients with CFDs, particularly in patients with hemophilias. The rarity and clinical heterogeneity of RBDs lead to significant diagnostic challenges and improper management. In this regard, multinational collaborative efforts are needed with the hope that can improve the management of patients with RBDs.

Venom Protein C Activators as Diagnostic Agents for Defects of Protein C System.

BACKGROUND: Protein C is a vitamin K dependent plasma zymogen. It inhibits clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converting the zymogen protein C (PC) of vertebrates into activated PC have been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. CONCLUSION: The fast-acting PC activator Protac® from Agkistrodon contortrix contortrix (Southern copperhead snake) venom has found a broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis.