RASopathies and hemostatic abnormalities: key role of platelet dysfunction

Abstract Background Bleeding anomalies have been reported in patients affected by Noonan syndrome. No study has been performed in patients with molecularly confirmed RASopathy. We aimed to characterize the frequency and types of bleeding disorders in patients with RASopathies and evaluate any significant association with laboratory findings. Patients and methods Forty-nine individuals (PTPN11, n = 27; SOS1, n = 7; RIT1, n = 3; SPRED1, n = 1; LZTR1, N = 3; RAF1, n = 2; BRAF, n = 4; MEK1, n = 1; MEK2, n = 1), and 49 age- and sex-matched controls were enrolled. The “Paediatric Bleeding Questionnaire Scoring Key” was administered to patients and families. Laboratory screening tests including clotting factors dosing, platelet count, Prothrombin Time and Partial Thromboplastin Time, were employed both in patients and controls to characterize the bleeding diathesis. A subgroup of 29/49 patients and 29/49 controls was also tested for platelet function. Results Regardless of the gene involved, pathological paediatric bleeding scores were recorded in 14/49 (28.5%) patients. Indeed, 7 were mutated in PTPN11, 3 in SOS1, 2 in RIT1, 1 in BRAF, and 1 in MEK1. Compared to patients with normal bleeding scores, those with pathologic bleeding score showed higher prevalence of splenomegaly (p = 0.006), prolonged aPTT (p = 0.04), lower levels of coagulation factor V (FV, p = 0.001), FVII (p = 0.003), FX (p = 0.0008) and FXIII (p = 0.002), higher vWAg (p = 0.04), and lower platelet sensitivity to Ristocetin (p = 0.001), arachidonic acid (AA) (p = 0.009) and collagen (p = 0.01). The presence of hematomas inversely correlated with factor V (p = 0.002), factor VII (p = 0.003), factor X (p = 0.002) and factor XIII (p = 0.004) levels, and directly correlated with platelet response to collagen (p = 0.02) and AA (p = 0.01). The presence of splenomegaly directly correlated with the presence of hematoma (p = 0.006), platelet response to Ristocetin (p = 0.04) and AA (p = 0.04), and inversely correlated with factor V levels (p = 0.03). Conclusions Patients with RASopathies and a bleeding tendency exhibit multiple laboratory abnormalities, including platelet-related disorders. Splenomegaly is frequently detected and might be a suggestive sign for qualitative platelet dysfunction. A comprehensive clinical assessment should be carried out at diagnosis, during the follow-up and before any surgical procedures. Since there is currently no consensus on management of bleeding complications, it is important that physicians closely monitor these patients.

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RASopathies and Hemostatic Abnormalities: Key Role of Platelet Dysfunction

10.21203/rs.3.rs-417615/v1 ◽

2021 ◽

Author(s):

Di Candia Francesca ◽

Marchetti Valeria ◽

Cirillo Ferdinando ◽

Di Minno Alessandro ◽

Rosano Carmen ◽

...

Keyword(s):

Coagulation Factor ◽

Screening Tests ◽

Factor Vii ◽

Bleeding Complications ◽

Bleeding Tendency ◽

Factor X ◽

Platelet Response ◽

Platelet Dysfunction ◽

Clotting Factors ◽

Laboratory Findings

Abstract Background: Bleeding anomalies occur in patients affected by Noonan syndrome. No study has been performed in patients with molecularly confirmed RASopathy. We aimed to characterize the frequency and types of bleeding disorders in patients with RASopathies and evaluate any significant association with laboratory findings. Patients and methods: Forty-nine individuals (PTPN11, n=27; SOS1, n=7; RIT1, n=3; SPRED1, n=1; LZTR1, N=3; RAF1, n=2; BRAF, n=4; MEK1, n=1; MEK2, n=1), and 49 age- and sex-matched controls were enrolled in the study. The “Paediatric Bleeding Questionnaire Scoring Key” was administered to patients and families. Laboratory screening tests including clotting factors dosing, platelet count, prothrombin time, and partial thromboplastin time, were employed both in patients and controls to characterize the bleeding diathesis. A subgroup of 29/49 patients and 29/49 controls was also tested for platelet function. Results: Regardless the gene involved, pathological bleeding scores were recorded in 14 (28.5%) patients. Among these, 7 were mutated in PTPN11 (26% of total cases with PTPN11 mutations), 3 in SOS1 (43%), 2 in RIT1 (67%), 1 in BRAF (25%), and 1 in MEK1. Compared to patients with normal bleeding scores, those with pathologic bleeding score showed higher prevalence of splenomegaly (p=0.006), prolonged aPTT (p = 0.04), lower levels of coagulation factor V (FV) (p = 0.001), factor VII (FVII) (p = 0.003), factor X (FX) (p = 0.0008) and factor XIII (FXIII) (p = 0.002), higher vWAg (p = 0.04), and lower platelet sensitivity to Ristocetin (p = 0.001), arachidonic acid (AA) (p = 0.009) and collagen (p = 0.01). The presence of hematomas inversely correlated with FV (p = 0.002), FVII (p = 0.003), FX (p = 0.002) and FXIII (p = 0.004) levels, and directly correlated with platelet response to collagen (p = 0.02) and AA (p = 0.01). The presence of splenomegaly directly correlated with occurrence of hematoma (p=0.006), platelet response to Ristocetin (p = 0.04) and AA (p = 0.04), and inversely correlated with FV levels (p = 0.03).Conclusions: Patients with RASopaties and a bleeding tendency exhibit multiple coagulation-related laboratory abnormalities, including platelet-related disorders. Splenomegaly is frequently detected and might be a suggestive sign for qualitative platelet dysfunction. A comprehensive clinical assessment should be carried out at diagnosis, during the follow-up and before any surgical procedures. Since there is currently no consensus on management of bleeding complications, it is important that physicians closely monitor these patients.

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The Thrombogram in Rare Inherited Coagulation Disorders: Its Relation to Clinical Bleeding

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613258 ◽

2002 ◽

Vol 88 (10) ◽

pp. 576-582 ◽

Cited By ~ 181

Author(s):

Raed Al Dieri ◽

Flora Peyvandi ◽

Elena Santagostino ◽

Muriel Giansily ◽

Pier Mannuccio Mannucci ◽

...

Keyword(s):

Lag Time ◽

Peak Height ◽

Factor Vii ◽

Clotting Factor ◽

Severe Bleeding ◽

Factor V ◽

Bleeding Tendency ◽

Factor X ◽

Factor Xi ◽

Factor Concentration

SummaryWe investigated the relation between clotting factor concentration, the parameters of the thrombin generation curve (the thrombogram) and the severity of clinically observed bleeding in patients with congenital deficiency of prothrombin (n = 21), factor V (n = 22), factor VII (n = 22), factor X (n = 10), factor XI (n = 7) and factor XII (n = 6). The parameters used were: area under the curve (endogenous thrombin potential, ETP), peak concentration of thrombin attained and lag time before manifest formation.Peak height and ETP varied linearly with the concentration of prothrombin. For the other factors these parameters hyperbolically approached to the 100% limit with increasing clotting factor concentration. Half normal ETP was seen at about the following concentrations: prothrombin (50%), factor V (1%), factor VII (2%), factor X (5%) and factor XI (1%). As a rule, the peak height was somewhat more sensitive to clotting factor decrease than the ETP was.In all the patients with severe bleeding symptoms the ETP was less than 20% of normal. Bleeding tendency was absent or mild in patients with an ETP of 30% or higher. This value (except for prothrombin) is already obtained at concentrations of clotting factor of 1%-2%, which corroborates the clinical observation that a severe bleeding tendency is only seen in severe clotting factor deficiencies (less than 1%). The one exception was a patient with factor VII deficiency and severe bleeding, who showed a normal ETP value, albeit with a decreased peak height and a prolonged lag-time.

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Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation

Blood ◽

10.1182/blood-2003-04-1199 ◽

2003 ◽

Vol 102 (12) ◽

pp. 4014-4020 ◽

Cited By ~ 32

Author(s):

Elisabetta Castoldi ◽

José W. P. Govers-Riemslag ◽

Mirko Pinotti ◽

Debora Bindini ◽

Guido Tans ◽

...

Keyword(s):

Thrombin Generation ◽

Clinical Phenotype ◽

Activated Protein C ◽

Coagulation Factor ◽

Factor Vii ◽

Factor V ◽

Factor X ◽

Fv Leiden ◽

Fvii Deficiency ◽

Thrombin Formation

Abstract We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting material-negative (CRM-) FVII defect (9726+5G>A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with normal FV or FV Leiden gradually decreased at increasing FVII concentration. We conclude that coinheritance of FV Leiden increases thrombin formation and can improve the clinical phenotype in patients with severe FVII deficiency. (Blood. 2003;102:4014-4020)

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Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽

10.1182/blood.v64.6.1220.bloodjournal6461220 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1220-1227

Author(s):

D Menache ◽

HE Behre ◽

CL Orthner ◽

H Nunez ◽

HD Anderson ◽

...

Keyword(s):

Animal Models ◽

Specific Activity ◽

Rabbit Model ◽

Coagulation Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Clotting Factors ◽

Coagulation Factor Ix

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

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A Review of Coagulation Abnormalities of Autoimmune Acquired Factor V Deficiency with a Focus on Japan

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0041-1740149 ◽

2021 ◽

Author(s):

Akitada Ichinose ◽

Tsukasa Osaki ◽

Masayoshi Souri

Keyword(s):

Coagulation Factor ◽

Nationwide Survey ◽

Autoimmune Disorder ◽

Antibody Detection ◽

Extensive Literature ◽

Factor V ◽

Bleeding Tendency ◽

Factor X ◽

Timely Initiation ◽

Fv Antibody

AbstractCoagulation factor V (or FV for the purpose of medical safety) is an essential cofactor of coagulation factor X in the common pathway of coagulation; severe FV deficiency leads to a bleeding tendency. Although both congenital and acquired FV deficiencies are widely recognized, FV deficiency also presents as an autoimmune disorder. A nationwide survey on autoimmune coagulation factor deficiencies (AiCFDs) conducted in Japan by our Japanese Collaborative Research Group identified 24 new patients with autoimmune FV deficiency (AiFVD) in the past 5 years. Furthermore, our extensive literature search confirmed that 177 AiFVD cases have been reported in previous articles published from Japan. Patients with AiFVD in Japan were predominantly men, with age similar to those with other AiCFDs. AiFVD was confirmed as a relatively mild type of bleeding diathesis, associated with lower mortality rate than that for AiFVD and other AiCFDs reported in previous studies. Patients with AiFVD had variable FV inhibitor titers and both neutralizing anti-FV autoantibodies and nonneutralizing counterparts. Although spontaneous resolution occurs in some patients, timely initiation of hemostatic and immunosuppressive therapies helps arrest the bleeding and eliminate anti-FV antibodies, resulting in a high cumulative recovery rate. Immunological anti-FV antibody detection is recommended to avoid missing AiFVD cases for the presence of nonneutralizing anti-FV autoantibodies. Further investigation is necessary to clarify the long-term prognosis and optimal management of AiFVD.

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Pathways of Blood Clotting Initiation by Cancer Cells

10.1055/s-0039-1687373 ◽

1979 ◽

Author(s):

N. Semeraro

Keyword(s):

Cancer Cells ◽

Blood Clotting ◽

Coagulation Factor ◽

Factor Vii ◽

Ehrlich Carcinoma ◽

Factor V ◽

Factor X ◽

Experimental Tumors ◽

Activate Coagulation Factor ◽

Available Information

Although available information indicates that cancer cells may activate blood coagulation, the precise mechanism remains still uncertain. A procoagulant with characteristics of tissue thromboplastin has been found in human benign and malignant tissues and in some experimental tumors. On the other hand it has been reported that extracts from malignant tissues directly activate coagulation factor X, due to the presence of a serine protease. We have investigated the procoagulscitic fluid. Cells from Lewis lung carcinoma (primary and metastasis), Ehrlich carcinoma ascites and JW sarcoma ascites were able to shorten markedly the recalcification time of normal, factor VIII and factor VII-deficient, not of factor X-deficient human plasma. The same cells did generate thrombin when mixed with a source of prothrombin and factor X, absorbed bovine serum (as a source of factor V), phospholipid and CaCl2.Cells from Sarcoma ISO ascites were completely inactive in both test systems. It was a included that cells from some experimental tumors, similarly to normal platelets, possess the capacity to directly activate coagulation factor X. This suggests the existence of an alternative “cellular” pathway in blood clotting initiation distinct from both the intrin sic and extrinsic mechanisms.(Supported by Italian CNR and NIH, NCI, USA).

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Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models

Blood ◽

10.1182/blood.v64.6.1220.1220 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1220-1227 ◽

Cited By ~ 47

Author(s):

D Menache ◽

HE Behre ◽

CL Orthner ◽

H Nunez ◽

HD Anderson ◽

...

Keyword(s):

Animal Models ◽

Specific Activity ◽

Rabbit Model ◽

Coagulation Factor ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Clotting Factors ◽

Coagulation Factor Ix

Abstract Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.

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The Formation of the Extrinsic Prothrombin Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654935 ◽

1961 ◽

Vol 05 (03) ◽

pp. 402-425 ◽

Cited By ~ 19

Author(s):

W Straub ◽

F Duckert

Keyword(s):

Tissue Factor ◽

Factor Vii ◽

Factor V ◽

Factor X ◽

Clotting Factors ◽

Second Stage ◽

Ca Ions ◽

Tissue Thromboplastin ◽

Congenital Factor

SummaryThe formation of the extrinsic activator of prothrombin conversion is investigated. We use the term “extrinsic activator” to avoid ambiguity which could arise when employing the name tissue thromboplastin.The experiments are carried out with purified clotting factors and congenital factor VII and X-deficient sera. Two steps can be distinguished. First, tissue factor and factor X react together as substrates in presence of Ca ions to form the extrinsic reaction product. The reaction is catalyzed enzymatically by factor VII. In the second stage, the reaction product and factor V (substrate) form the extrinsic activator which in turn can convert the prothrombin to thrombin.

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Effect of Neutral Proteases of Human Granulocytes on Isolated Clotting-Factors

10.1055/s-0039-1689546 ◽

1975 ◽

Author(s):

W. Schmidt ◽

R. Egbring ◽

K. Havemann ◽

H. Beeser

Keyword(s):

Coagulation Factor ◽

Coagulation Factors ◽

Factor Vii ◽

Factor V ◽

Clotting Factors ◽

Human Granulocytes ◽

Rapid Destruction ◽

Weak Activity ◽

Moderate Effect ◽

Antigen Antibody

To examine whether direct proteolysis of coagulation factors may play a role in patients with so called consumption coagulopathy, an elastase-like and a chymotrypsin-like neutral protease isolated from human granulocytes were investigated for their influence on several purified clotting factors. The elastaselike protease induced a rapid destruction of fibrinogen, factors II, VIII, XII and XIII activity, whereas a moderate effect on factor V and VII activity was observed. The chymotrypsin-like enzyme showed a rapid inactivation of factor VIII, moderate effect on factor VII and XIII and only a weak activity against fibrinogen, factor II and XIII. Incubation of factor V with both enzymes leads to a transitory activation. In spite of the presence of a high antiprotease potential in plasma, addition of the elastase-like enzyme to normal plasma resulted in an activation of several coagulation factors. As it has been shown that the proteases are activity released from granulocytes in presence of antigen-antibody complexes, endotoxin and polynucleotides, the results given above together with the appearence of granulocytic proteases in the plasma of patients with acute leucemia and septicemia suggest that in certain types of coagulation factor deficiencies direct proteolysis rather than consumption of clotting factors due to dissiminated intravascular coagulation may be operational.

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Cloning and Characterization of the Murine Coagulation Factor X Gene

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613901 ◽

2000 ◽

Vol 83 (05) ◽

pp. 732-735 ◽

Cited By ~ 2

Author(s):

Adrian Cooper ◽

Zhong Liang ◽

Francis Castellino ◽

Elliot Rosen

Keyword(s):

Factor Viii ◽

Coagulation Factor ◽

Factor Ix ◽

Coagulation Factor Viii ◽

Start Codon ◽

Factor Vii ◽

Factor V ◽

Factor X ◽

Coagulation Factor Vii ◽

Coagulation Factor V

SummaryThe gene encoding murine coagulation factor X (fX) was isolated and characterized from a λFIX II library generated from murine genomic DNA. The 20130 bp sequence contains 18049 nucleotides that extend from the initiating methionine to the polyadenylation site. 1056 nucleotides 5’ of the start codon were determined and contain putative start sites for the FX mRNA as well as sites for binding of putative transcription factors. The sequence extends 1024 3’ of the polyadenylattion site.The gene contains 8 exons and 7 introns which were determined by comparing the mouse FX cDNA and gene sequences. The exonic structure of the gene is similar to that of the other mammalian vitamin K-dependent serine proteases of the coagulation system. These include an exon encoding the prepropepetide, the gladomain, a short helical stack, two exons for the two EGF domains, the activation pepetide, and two exons encoding the serine protease domain. The 5’ sequence of the mouse FX gene overlaps with the 3’ region of the FVII gene indicating that the murine FVII and FX gene are arranged in a head to tail arrangement as they are in humans. Abbreviations: fVII, coagulation factor VII; fIX, coagulation factor IX; fX, coagulation factor X; PC, Protein C; fV, coagulation factor V; fVa, activated coagulation factor V; fVIII, coagulation factor VIII; fVIIIa, activated coagulation factor VIII.

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