Mild Bleeding Tendency Research Articles

Myocardial infarction in two cousins heterozygous for ASN41HIS autosomal dominant variant of Bernard–Soulier syndrome

Bernard-Soulier Syndrome is characterized by thrombocytopenia with large platelets and defective aggregation to ristocetin. The bleeding tendency is variable but may be severe. The syndrome is due to genetic defects of the GPIb-V-IX complex and it has been maintained to be protective from thrombotic events. Here we present the first two cases of documented M.I. in two cousins, heterozygous for the Arg41His mutation which is responsible for a dominant form of Bernard-Soulier Syndrome. In one of the two patients an aneurysm of the aorta was also present. The patients had a mild bleeding tendency which was severely aggravated by treatment with antiplatelet drugs. These clinical observations are in contrast with experimental studies which demonstrate that Bernard-Soulier-like strains of mice show a decreased thrombus generation in several experimental settings.

Congenital FVII Deficiency and Pulmonary Embolism

Fourteen patients with congenital factor VII (FVII) deficiency were reported to have had pulmonary embolism. All patients were type 2 defects with variably low activity but normal or near-normal antigen. Concomitant deep vein thrombosis was present in 7 instances. The majority of patients had no or only a mild bleeding tendency. Associated prothrombotic risk factors were present in 11 patients (old age, surgery, substitution therapy with prothrombin complex, plasma-derived or activated FVII concentrates). Pulmonary embolism was usually moderate or severe. In 2 cases, it was fatal. Only 4 patients were studied by means of molecular biology techniques. The Arg304Gln mutation was found in 5 of the 8 alleles. Heparin and Coumadin together with adequate substitution therapy were carried out in 5 patients with satisfactory results. The FVII deficiency does not grant a sure protection from venous thromboembolism.

A cluster of factor XI-deficient patients due to a new mutation (Ile 436 Lys) in northeastern Italy*

A new mutation (Ile 436 Lys) was found in a cluster of patients in northeastern Italy. The mutation was present in five patients at the homozygote level and in one patient as a compound heterozygote with an already known mutation namely Glu 117 stop. All these patients showed a mild bleeding tendency mainly associated with deliveries or surgery. The first two patients were two sisters, and their parents were consanguineous. The third patient was the only homozygote in the family, and parents apparently were not consanguineous. The fourth and fifth patients were a brother and a sister, and in this case too, parents were not consanguineous. The sixth patient, a compound heterozygote, negated also the existence of consanguinity between his parents. There were also seven heterozygotes among the family members of the patients homozygous for this new mutation (Ile 436 Lys). Finally, there were two heterozygotes for the Glu 117 stop mutation in the family of the sixth patient. The heterozygotes, regardless of the mutation, were asymptomatic. The Ile436Lys mutation is characterized by low factor XI activity and antigen, namely is a cross-reaction material negative form. Molecular modeling indicates that the Ile436Lys mutation causes a large conformational change within the 432-442 loop. No relation could be traced among the different families; however, all their ancestors were autochthonous of the same two small towns. Furthermore, no Jewish ancestry could be found. The close geographical area in which all these patients were found and the absence of the same mutation in the general population of the area strongly suggests a founder effect and that the mutation is responsible for the defect. The compound heterozygosis with the Glu 117 stop mutation, common among Jews, was not surprising because of the past strict ties of the Republic of Venice with the Middle East.

Clinical and laboratory features of 103 patients from 42 Italian families with inherited thrombocytopenia derived from the monoallelic Ala156Val mutation of GPIb (Bolzano mutation)

Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients. Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin. We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases. Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.

Recent advances in the understanding and management of MYH9‐related inherited thrombocytopenias

MYH9-related disease (MYH9-RD) is one of the most frequent forms of inherited thrombocytopenia. It is transmitted in an autosomal dominant fashion and derives from mutations of MYH9, the gene for the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia with mild bleeding tendency and may develop kidney dysfunction, deafness and cataracts later in life. The term MYH9-RD encompasses four autosomal-dominant thrombocytopenias that were previously described as distinct disorders, namely May-Hegglin Anomaly, Sebastian, Fechtner and Epstein syndromes. Thrombocytopenia is usually mild and derives from complex defects of megakaryocyte maturation and platelet formation. It is easily diagnosed, in that the presence of giant platelets in peripheral blood raises the suspicion of MYH9-RD and a simple immunofluorescence test on blood films confirms the diagnostic hypothesis. However, genotype/phenotype correlations have been recognized and mutation screening is therefore required to define the risk of acquiring extra-haematological defects. Results of a small clinical study suggested that a non-peptide thrombopoietin mimetic might greatly benefit both thrombocytopenia and bleeding tendency of MYH9-RD patients.

MYH9 Related Platelet Disorders - Often Unknown and Misdiagnosed

MYH9 related platelet disorders are a relatively rare cause of thrombocytopenia. Located on chromosome 22, the MYH9 gene encodes the motorprotein non-muscular myosin heavy chain IIA (NMMHCIIA). Heterozygous defects in this gene lead to 4 different autosomal dominant syndromes namely May-Hegglin anomaly, Epstein syndrome, Fechtner syndrome and Sebastian platelet syndrome. All 4 syndromes are characterized by macrothrombocytopenia and a mild bleeding tendency. Depending on the position of the causative mutation within the gene, the risk increases for syndromic manifestations such as renal failure, hearing loss and pre-senile cataract. Mutations in the neck region of the NMMHCIIA protein are more likely associated with these comorbidities than mutations in the N- or C-terminal part of the gene. MYH9 related platelet disorders should be excluded in patients with chronic thrombocytopenia and large platelets. Most sensitive for diagnosis/exclusion are immunofluorescence studies using a blood smear. The biggest risk for these patients is ineffective but potentially harmful treatment based on the misdiagnosis of immune thrombocytopenia. This review provides a workflow for diagnosis and treatment of MYH9 related thrombocytopenia.

Sideroblastic Anemias Associated with Marked Thrombocytosis, the Role of JAK2 (V616F) Mutation (exon 6 and 12)

AbstractAbstract 5135The refractory anemia with ringed sideroblasts (RARS) and multilineage dysplasia is often associated with marked thrombocytosis; the research of JAK2 (V617F) mutant alleles was positive, JAK2 mutation is present in patients with polycythaemia vera (70%-95%), essential throbocythemia (32%-58%), CIMF (36%-50%), resulting in a valine to phenylalanine substitution at position 617; the mutation increases the kinase activity of JAK2 and the auto-phosphorylation and hyperactivation of EPOR and TPOR these two causing eritroid cells hyperplasia, the increase of dysgranulocytopoiesis with small hypolobulated dysplastic form and megakaryocytopoiesis with cluster of giant cells and enhanced risk for thrombosis greater than 30% or recurrent thrombosis greater than 20%. Dysgranulocytopoiesis features includes hypogranulation of myeloid cells, pseudo Pelger form, ringed sideroblastosis, thrombocytosis (>600.000/nL) with refractory cytopenia, and signficantly higher levels of PF4.We have studied 8 patients with RARS–T, and we have found that JAK2 mutation is present in 5 out of 8 patients; these patients had a significantly higher white cells count, an increase of the tyrosin-kinase 2, and an enhanced activation of downstream transcription factors such as STAT-3 and STAT-5 A/B signalling cytokines that induce FOXP3 up regulation in activated CD4+, CD25+ regulatory T cells. Several defects of aggregation are present and deficiency of ATIII, PC, PS, splenomegaly were associated with a mild bleeding tendency. Disclosures:No relevant conflicts of interest to declare.

Prevalence of Suspected Collagen Disorders In the Adult Bleeding Disorder Clinic.

AbstractAbstract 1399 Background:Disorders of collagen may be associated with a mild bleeding tendency because of the interaction of collagen with von Willebrand factor (VWF) and platelets required during primary hemostasis and generalized soft tissue fragility. Therefore, collagen disorders may be unrecognized contributors to existing disorders of primary hemostasis. Synergism between these entities could produce a more significant bleeding tendency than predicted based on laboratory abnormalities alone. Symptomatic joint hypermobility (SJH) is found in over 90% of collagen disorders and can be objectively measured as a surrogate marker of a potential collagen disorder. Objective:To determine the prevalence of suspected collagen disorders in a cohort of adult subjects with mucocutaneous inherited bleeding disorders in comparison to healthy controls using SJH as a marker for a potential collagen disorder. Methods:Fifty-five consecutive subjects, ≥ 16 years of age, attending the Adult Bleeding Disorders (BD) Clinic with a history of von Willebrand disease, platelet function disorder or undefined bleeding disorder and 60 controls (age and sex-matched) were enrolled between June 2008 and February 2010. All subjects were assessed for: i) Generalized joint hypermobility, defined using the Beighton score whereby a score of ≥ 4 out of 9 is considered positive; ii) SJH, defined using the major Brighton criteria which is a Beighton score of ≥ 4 AND arthralgia for longer than 3 months in 4 or more joints; and, iii) Bleeding score (BS), using the Condensed MCMDM1-VWD, with a positive score defined as ≥ 4 for both sexes. CBC, blood group, VWF:Ag, VWF:RCo, FVIII:C and closure times were collected prospectively in the control group and retrospectively in the clinic subjects in addition to bleeding time and platelet aggregation studies. Results:The prevalence of SJH with a positive bleeding score was 24% in the BD clinic and 1.6% in controls (see table).Seventy-seven percent of SJH subjects (10/13) in the BD clinic had a prior personal or family history of Ehlers-Danlos Syndrome, other Joint Hypermobility Syndrome or Osteogenesis Imperfecta (OI). Only the OI subject had prior clinical documentation of a collagen disorder. There was no statistical difference in laboratory measures of hemostasis or bleeding score in the BD clinic subjects with or without SJH. Of interest, mean bleeding time in the BD clinic subjects (available in 38 subjects) was 10.5 minutes in the SJH group and 10 minutes in the non-SJH group suggesting that bleeding time is not a good screening test for the presence of abnormal collagen. Isolated joint hypermobility (Beighton score of ≥ 4) not associated with arthralgia was common in both groups (40% and 30% respectively) in this predominately female population of subjects with inherited mucocutaneous bleeding. Conclusions:SJH suggesting a collagen disorder is common and often unrecognized in the BD clinic as a potential contributor to the bleeding symptoms. Asymptomatic joint hypermobility is common in this predominately female population and not an adequate screening tool for a potential collagen disorder. A prolonged bleeding time did not predict the presence of SJH in this cohort. Further study is required to confirm the nature of collagen disorders in the BD clinic population and the relationship to bleeding manifestations. Disclosures:No relevant conflicts of interest to declare.

大動脈瘤によるChronic DICを呈した高齢腎不全患者の2例―ヘパリン持続皮下注射療法の有効性も含めて

We describe here two cases of chronic disseminated intravascular coagulation(DIC) secondary to aortic aneurysms. The patients were 78- and 84-year-old males, who visited our hospital to receive hemodialysis therapy for chronic renal failure probably due to nephrosclerosis. They had mild bleeding tendency and thrombocytopenia(< 10 x 10(4)/microliter). Coagulation test revealed the findings of chronic DIC in both patients, and computed tomography showed abdominal and thoracoabdominal aortic aneurysms with mural thrombi, respectively. In one patient, subcutaneous hemorrhage after vascular access surgery had continued for a month. However, the hemorrhage and swelling of the limb disappeared after continuous subcutaneous heparin infusion(CSHI) therapy in a daily dose of 10,000-14,000 unit. These findings suggest that chronic DIC secondary to aortic aneurysm should be considered when bleeding tendency and thrombocytopenia are observed in aged patients, and that CSHI is the choice of therapy for the bleeding tendency of chronic DIC.

Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile.

Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. This case-control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet-rich plasma than controls (P < 0.05). By multivariate logistic regression, only non-null mutations were confirmed as an independent predictor of a mild clinical phenotype. These results indicate that non-null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet-rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype.

Characterisation of a novel nonsense mutation in FGG (Fibrinogen Poznan) causing hypofibrinogenaemia with a mild bleeding tendency

Characterisation of a novel nonsense mutation in FGG (Fibrinogen Poznan) causing hypofibrinogenaemia with a mild bleeding tendency -

Platelet dysfunction and inhibition of multiple electrode platelet aggregometry caused by penicillin

Beta-lactam antibiotics, e.g. penicillin, may inhibit platelet function and lead to reduced response in light transmission aggregometry and adhesion. However, influence on platelet function tests more commonly used in clinical practice, such as multiple electrode platelet aggregometry (MEA), have not been described so far. We report a case of a patient with local streptococcus infection. Treatment with penicillin resulted in mild bleeding tendency after 3 days. While coagulation parameters were normal, assessment of platelet function by MEA revealed strong platelet inhibition of both ADP and arachidonic acid induced platelet aggregation comparable to normal responders to antiplatelet therapy. Change of antibiotic regime resulted in recovery of platelet function. Thus, penicillin therapy may impact on platelet function and consecutively commonly used platelet function assays, e.g. MEA.

Untersuchungen zur Hämostase bei Kindern unter Valproatbehandlung

SummaryCoagulation parameters were determined in children with valproic acid mono- and valproic acid-lamotrigin combination therapy. Patients, methods: Monotherapy group (n = 22; mean age: 10.5 years) was compared to combination therapy (n = 7; 12.9 years) and a control group (n = 22; 8.7 years). The following parameters were measured: aggregation and ATP-release in whole blood (ADP: 20 μmol/l, collagen: 1 μg/ml, thrombin: 0.5 U/ml), PFA-100® closure times (CT), blood cell counts, global tests, VWF : Ag, VWF : CBA, factors VIII and XIII as well as fibrinogen. Bleeding symptoms were evaluated by using a questionnaire. Results: For ADP- and collageninduced aggregation as well as for ATP release no significant differences between the groups were detected. The combined therapy group showed significantly prolonged CT. Von Willebrand disease was not detected in any of the patients. The platelet count was significantly decreased in the monotherapy group. In six children a mild bleeding tendency was observed, mostly epistaxis. Conclusion: A clinically relevant influence of valproic acid on haemostasis was found only in few cases. However, before surgical procedures an extended coagulation diagnostics is recommended in patients with valproic acid therapy.

Abnormal hemostasis in a knock-in mouse carrying a variant of factor IX with impaired binding to collagen type IV.

Factor IX binds to collagen type IV, but this binding has no known consequence. To determine the effect of reduced binding of FIX to collagen IV. We constructed and characterized 'knock-in' mice containing the mutation lysine 5 to alanine (K5A) in the Gla domain of their FIX. The K5A mutation dramatically reduced the affinity of FIX for collagen type IV, but had no measurable effect on platelet binding, phospholipid binding, or in vitro clotting activity. However, K5AFIX mice had a mild bleeding tendency, despite their in vitro clotting activity being normal. Hemostatic protection from delayed rebleeding was intermediate between wild-type and hemophilia B mice (which had no detectable clotting activity); moreover, survival of K5A FIX mice after nascent clot removal was dramatically improved as compared with hemophilia B mice. Importantly, there was no detectable difference between K5AFIX and wild-type mice in either a laser-induced thrombosis model or the chromogenic FIX activity assay. In contrast, after ferric chloride injury, which exposes collagen IV as well as other basement membrane proteins, intravital microscopy revealed that vessel occlusion was significantly slower in K5AFIX mice than in wild-type mice. Our results indicate that the FIX molecule with decreased affinity for collagen IV has altered hemostatic properties in vivo and that the binding of FIX to collagen IV probably plays a significant functional role in hemostasis.

Interpatient Phenotypic Inconsistency in Severe Congenital Hemophilia: A Systematic Review of the Role of Inherited Thrombophilia

It is well known that the clinical phenotype of hemophilia may vary greatly among patients with the same apparent level of coagulation factor and the same genetic mutation. Thus, patients with severe hemophilia may experience a severe phenotype or only a milder bleeding tendency, suggesting some other moderating influence. To elucidate the mechanism of this heterogeneity, some investigators have recently suggested that inherited thrombophilic factors may play a role in the milder clinical presentation of severe hemophilia. In this review, we summarize current knowledge with respect to the modulation of the clinical phenotype of severe hemophilia by prothrombotic genetic risk factors. Although the published literature seems to indicate a protective effect for the coinheritance of factor V Leiden, the limited data available do not permit any firm conclusions. Further trials on a large population of patients are needed to establish the role of genetic thrombophilia in the phenotypic expression of severe hemophilia.

Absence of platelet-dependent fibrin formation in a patient with Scott syndrome

To gain insight into the contribution of platelet-dependent thrombin formation in haemostasis and thrombosis, we investigated under flow conditions the haemostatic functions of platelets from a patient with Scott syndrome. Scott platelets are characterised by a diminished platelet-dependent thrombin generation. Thrombin generation was determined by calibrated automated thrombography and flow-based experiments were performed to reveal collagen-mediated platelet activation and fibrin deposition. Our studies indicate that adherent Scott platelets do not differ from control platelets in the formation of stable platelet aggregates under static and flow conditions. While for adherent control platelets a shape change, e.g. balloon formation, and externalisation of phosphatidylserine (PS) is associated with an increase in intracellular calcium concentration, this is not the case for Scott platelets. The calcium-induced morphological changes in control platelets are accompanied with a diminished recruitment of free flowing platelets. Scott platelets, not showing a calcium-induced shape change, also lost the ability to recruit free flowing platelets. These findings rebut the hypothesis that the mild bleeding tendency of Scott syndrome patients is due to a preserved adhesive activity of patient's platelets. Perfusion of tissue factor (TF)-activated control blood over immobilised collagen results in the formation of fibrin fibers that radiate from platelet aggregates. Although platelet aggregates were also observed after perfusion with TF-activated Scott blood, fibrin deposition was not observed. In conclusion, our findings indicate that platelet adhesion and spreading on a collagen matrix in the absence of fibrin formation is sufficient to sustain haemostasis under non-traumatic conditions.

Increased Thrombin Generation in Severe Hemophiliacs with Mild Clinical Phenotype

Introduction: Some severe hemophiliacs (FVIII/FIX<1%) exhibit a mild bleeding tendency, but the basis for this clinical heterogeneity is poorly understood. This study investigated the relationship between the values of endogenous thrombin potential (ETP) and clinical phenotype in severe hemophiliacs. The impact of FVIII/FIX gene mutations and thrombophilic polymorphisms was also evaluated. Methods: severe hemophiliacs older than 18 years without inhibitor history and treated on demand were eligible. Mild bleeders (MB) and severe bleeders (SB) were defined as follows: spontaneous bleeding episodes per year ≤2 (MB) or 25 (SB) and concentrate consumption <500 (MB) or >2000 (SB) IU/Kg/year. Patients who did not fit these criteria were considered as intermediate bleeders (IB). FVIII was measured by chromogenic assay and ETP was measured in platelet-rich plasma after addition of tissue factor. Results: 22MB, 22SB and 28IB were enrolled. MB had lower clinical and radiological scores when compared with both IB and SB (p<0.005). MB showed an older age at first bleed compared to SB (p < 0.005) and p for trend among the 3 groups was also significant (p < 0.05). The prevalence of severe FVIII/FIX gene defects (null mutations) was lower and ETP values were higher in MB compared with both IB and SB (p<0.05; table 1). Conclusions: our results indicate an extremely low prevalence of null mutations in severe hemophiliacs with mild bleeding diathesis. The measurement of thrombin generation in platelet-rich plasma may allow to identify this subgroup of patients, not otherwise distinguishable by conventional functional assays.SB (#22)IB (#28)MB (#22)pAge (yr)38 (21–76)38 (23–62)32 (22–73)NSAge 1st bleed (yr)1 (0–4)2 (0–6)3 (1–10)< 0.005Bleeding episodes/yr36 (25–60)10 (3–20)0 (0–2)< 0.0005Factor use (IU/Kg/yr)2207 (2040–8696)1068 (207–2400)60 (25–487)< 0.0005Clinical score18 (10–35)10 (0–34)3 (0–17)< 0.005Pettersson score44 (14–62)28 (0–48)17 (3–40)< 0.0005Null mutations (%)59706< 0.005PTG20210A (%)075NSFV Leiden (%)570NSMedian ETP (nM)414478850< 0.05

An unusual clinical presentation of factor XIII deficiency and issues relating to the monitoring of factor XIII replacement therapy

Congenital factor XIII deficiency is a very rare bleeding disorder. Patients with severe FXIII deficiency usually exhibit severe bleeding diatheses. Factor XIII is also involved in maintaining pregnancy, and women with factor XIII deficiency have a high risk of spontaneous abortions. We report here the case of a patient with a mild bleeding history before her pregnancy but who had three spontaneous haemorrhagic miscarriages. The patient was homozygous for G 501 R mutation of the factor XIII A subunit gene. We also detected a coinherited heterozygous factor V Leiden mutation, probably leading to a milder bleeding tendency. The patient had successful factor XIII replacement therapy throughout her fourth pregnancy. The efficacy of the factor XIII infusions was monitored using thromboelastometry and routine factor XIII measurements. This case report shows that factor XIII deficiency should be ruled out in patients with recurrent fetal loss but with a normal miscarriage workup, even in the absence of a history of severe bleeding since childhood. We also showed that thromboelastometry could be a valuable tool for the monitoring of factor XIII replacement therapy.

Thrombin-generating capacity in patients with von Willebrand's disease

on Willebrand's disease (VWD) is the most common hereditary bleeding disorder. Its severity can be classified on the basis of von Willebrand factor (VWF) and factor VIII (FVIII) plasma levels and according to the clinical relevance of bleeding episodes. However, patients with very low VWF activity may exhibit a mild bleeding tendency. The basis for this heterogeneous clinical expression of the deficit is still poorly understood. We investigated the relationship between thrombin generation and levels of factor VIII, VWF and clinical bleeding tendency. Thrombin generation was measured in platelet-rich (PRP) and platelet-poor plasma (PPP) from 53 patients with VWD. We observed a statistically significant higher risk of bleeding in patients with a low thrombin peak in PRP (OR=14.5; 95% CI=5-41.3). Similar results were found in PPP (OR=8.71; 95% CI=3.4-22.3). Two parameters of the thrombin generation curve, peak height and thrombin generation speed (slope), correlated significantly with VWF:RCo and FVIII levels both in PPP and in PRP. Regression analysis showed that thrombin generation was mainly dependent on plasma FVIII activity. Our results suggest that the thrombin generation test, in combination with routine FVIII and VWF measurements, could be of interest in the assessment of the individual bleeding risk in patients with VWD.

In Vitro and In Vivo Characterization of a Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FXRoma.

The complex and dynamic interplay between the intrinsic and extrinsic pathways of blood coagulation is incompletely understood. The mediator of prothrombin cleavage, Factor X (FX), plays a pivotal role as part of both the extrinsic and intrinsic tenase complexes. Moreover, the existence of naturally occurring Factor X mutations that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., 1988), is due to a missense mutation (Thr318←Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3% wild-type) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50% wild-type) in the extrinsic pathway as measured by PT. We chose to generate a mouse model of FX asymmetric activation to further probe the extrinsic-intrinsic pathway physiological relationship in hemostasis and thrombosis. For this, we used both an in vitro and an in vivo approach. We first constructed and purified the mouse homolog of FXRoma (mFXRoma) as well as wild-type mFX. Using a clotting-based assay, mFXRoma exhibited intrinsic and extrinsic activity comparable to that reported for the human mutation (5% and 18%, respectively). The reduced intrinsic and extrinsic activity of mFXRoma was not due to a secretion defect, based on Western blot analysis of supernatant and cell extracts from mFXRoma and mFX stably-transfected human embryonic kidney (HEK-293) cell lines. Mice homozygous for the analogous mutation (Thr315←Met) in exon 8 of the murine FX gene were generated by using a plug-and-socket approach. This resulted in the endogenous mFX exon 8 sequence being replaced with the mutated one, thus affording gene expression under the endogenous promoter. Analysis of mFXRoma homozygous mice showed a 6.4% and 19.2% intrinsic and extrinsic activity relative to wild-type littermates, respectively, confirming our in vitro data. The reduced activity in these mice resulted in a slight reduction in levels of the thrombin-antithrombin (TAT) complex. To determine any physiological defect of this mutation on the two pathways of coagulation, we performed two hemostatic challenges of the macrocirculation (tail clip and FeCl3-induced thrombus formation). In the tail-clip assay, blood loss showed no statistical difference between wild-type (n=5) and mFXRoma (n=6) mice. In contrast, following FeCl3-induced injury on the carotid artery (larger vessel diameter that in the tail), mFXRoma mice (3/3) failed to result in vessel occlusion (up to 30 min of observation), whereas wild-type littermates showed stable vessel occlusion (3/4) within ∼6 min of FeCl3 application. Although the type of injury was different, these data suggest that an impeded intrinsic activity of FX does not appear to affect hemostasis of the macrocirculation at relatively small diameter vessels but is essential for thrombus formation in large diameter vessels, and a relatively normal extrinsic activity does not compensate for this defect. This mouse model will aid in determining the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway of coagulation.