Sabrina Gavasso

X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua)

We report a case of juvenile thrombophilia associated with a substitution of leucine for arginine at position 338 (R338L) in the factor IX gene (factor IX-R338L). The level of the mutant factor IX protein in plasma was normal, but the clotting activity of factor IX from the proband was approximately eight times the normal level. In vitro, recombinant factor IX-R338L had a specific activity that was 5 to 10 times as high as that in the recombinant wild-type factor IX. The R338 substitution causes a gain-of-function mutation, resulting in factor IX that is hyperfunctional.

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in 4 patients with severe congenital FV deficiency (3 with plasma FV clotting activity [FV:C] < 1%). When triggered with tissue factor (TF) concentrations up to 50pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1 to 5pM of TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca(2+)-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.

Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency

Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n = 11; 4.0 +/- 1.0 ng/mL free TFPI) than in controls (n = 20; 11.5 +/- 4.8 ng/mL), while persons with partial FV deficiency had inter-mediate levels (n = 16; 7.9 +/- 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding.

Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism.

BACKGROUND Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Microparticles (MPs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play a role in VTE, although evidence of this association is still limited. OBJECTIVES To evaluate the hypothesis that elevated numbers of endothelial (EMPs), platelets (PMPs), and Tissue Factor-bearing MPs (TF(+)MPs) in plasma may contribute to cancer-associated thrombosis. PATIENTS/METHODS EMPs, PMPs and TF(+)MPs plasma levels were measured in 90 consecutive patients (cases) referred to our Department (30 with a first episode of unprovoked VTE; 30 with active cancer; 30 with a diagnosis of acute VTE associated with active cancer), and in a group of 90 healthy subjects (controls). MPs analyses were performed by flow-cytometry (Cytomics FC500). RESULTS Cases showed statistically significant higher (mean ± SD) circulating EMPs and PMPs plasma levels (920 ± 341 and 1221 ± 413 MP/μL, respectively) than controls (299 ± 102 and 495 ± 241 MP/μL; p<0.005). Moreover cancer patients (with and without VTE) showed higher (mean ± SD) TF(+)MPs (927 ± 415 MPs/μL) than controls (204 ± 112 MPs/μL; p<0.001). The subgroup of cancer patients plus VTE showed statistically significant higher TF(+)MPs plasma levels (1019 ± 656 MPs/μL) than cancer patients without VTE (755 ± 391 MPs/μL, p = 0.002). Multivariate analysis failed to show a significant association between elevated TF(+)MPs and VTE in cancer patients. CONCLUSIONS Our results suggest that MPs might be an important intermediate in the cascade of cellular injury and vascular dysfunctions underlying the process of thrombosis, particularly in cancer. Further clinical investigations are needed to confirm the precise role of MPs in predicting hypercoagulable state in patients with cancer.

Prothrombin fragment 1+2 and thrombin-antithrombin complex levels in patients with inherited APC resistance due to factor V Leiden mutation.

Inherited activated protein C (APC) resistance is a newly described pathological condition associated with familial thrombophilia. A recent report on a family with APC resistance showed increased levels of prothrombin fragment 1+2 (F1+2) in the affected individuals. No data concerning thrombin–antithrombin complex (TAT) levels in patients with inherited APC resistance are presently available.  The aim of this study was to assess the plasma levels of F1+2 and TAT in patients with inherited APC resistance due to factor V (F.V) Leiden mutation and to evaluate F1+2 and TAT levels in symptomatic and asymptomatic patients with the defect (‘carriers’) as compared to their family members having no evidence of F.V Leiden mutation (‘non‐carriers’).  One hundred and twenty‐nine individuals belonging to 30 families with inherited APC resistance due to F.V Leiden mutation were studied. F1+2 and TAT levels were determined using two commercially available ELISA kits and cut‐off values were defined as the higher limits of normal ranges obtained in healthy volunteers.  Out of the 129 family members investigated, 36 were non‐carriers, 85 were heterozygous and eight homozygous for F.V Leiden mutation. Thrombosis had occurred in 2/36 (6%) non‐carriers, in 36/85 (42.3%) heterozygous and in 5/8 (63%) homozygous. Median levels of F1+2 and TAT were above cut‐off values in carriers, whereas they were below in non‐carriers.  An overall percentage of 68.8% of carriers exhibited F1+2 levels above the cut‐off value as compared to 38.9% of non‐carriers. For TAT, an overall percentage of 63.4% of carriers presented with levels above the cut‐off compared with 28% of non‐carriers.  In conclusion, patients with inherited F.V Leiden mutation may exhibit increased levels of F1+2 and TAT. There are no differences in F1+2 and TAT median levels among symptomatic and asymptomatic carriers. The percentage of carriers of F.V Leiden with levels of F1+2 and TAT above cut‐off appears to be higher than that found in other clotting inhibitors defects and in this respect the defect might be considered different. However, these findings and the presence of a high percentage of non‐carriers presenting with increased F1+2 and TAT levels may suggest the possible coexistence in these families of other unknown defects predisposing to thrombosis.

Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5–10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non‐carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n = 178) or asymptomatic (n = 99) non‐carriers. The percentage of subjects with prothrombin levels above cut‐off values of 1.15 u/ml or 1.30 u/ml was significantly higher in carriers of the prothrombin variant as compared to non‐carriers, regardless of a previous thrombosis. However, among non‐carriers the percentage of those with prothrombin levels above cut‐off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non‐carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.

Increased anticoagulant response to low-molecular-weight heparin in plasma from patients with advanced cirrhosis

Summary.  Introduction:  Cirrhotic patients may present thrombotic complications that warrant anticoagulant therapy. However, the efficacy of low‐molecular‐weight heparin (LMWH) in this clinical setting is still unclear.

Differential effects of high prothrombin levels on thrombin generation depending on the cause of the hyperprothrombinemia.

Summary.  Background: Hyperprothrombinemia, resulting from the prothrombin G20210A mutation or other causes, is associated with activated protein C (APC) resistance and increased thrombosis risk. When high prothrombin levels are a result of increased hepatic biosynthesis, these effects may be counteracted by concomitantly increased levels of the anticoagulant factors (particularly protein S). Differently, in prothrombin G20210A carriers only prothrombin levels are elevated. Objective: To investigate whether prothrombin G20210A carriers have a more severe hypercoagulable state than non‐carriers with comparable prothrombin levels. Patients/methods: Coagulation factor levels, thrombin generation (Calibrated Automated Thrombogram in the presence and absence of APC) and APC resistance were measured in normal (n = 132), heterozygous (n = 167) and homozygous (n = 3) individuals. Results: Prothrombin levels, thrombin generation and APC resistance were higher in carriers of the prothrombin G20210A mutation (especially those who had experienced venous thrombosis) than in non‐carriers, whereas protein S and antithrombin levels were similar among genotype groups. Because individuals with high prothrombin levels in the absence of the prothrombin G20210A mutation tend to have all liver‐synthesized factors elevated, carriers of the mutation had lower protein S and antithrombin levels than non‐carriers with equally high prothrombin levels. Accordingly, they also generated more thrombin and showed a tendency toward higher APC resistance. Analogous effects, but less pronounced, were observed in homozygotes for the prothrombin A19911G polymorphism, which also upregulates prothrombin levels.Conclusions: Individuals with hyperprothrombinemia as a result of prothrombin gene mutations generate more thrombin and tend to be more APC‐resistant than individuals with comparable prothrombin levels because of other causes.

Similar hypercoagulable state and thrombosis risk in type I and type III protein S-deficient individuals from families with mixed type I/III protein S deficiency

Background Protein S, which circulates in plasma in both free and bound forms, is an anticoagulant protein that stimulates activated protein C and tissue factor pathway inhibitor. Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned. Design and Methods Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, or having type I or type III deficiency according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals). Results Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, tissue factor pathway inhibitor and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and two large deletions were identified in the genetically characterized families. Conclusions Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased risk of thrombosis. These findings may, however, be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

Background. Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. Methods. To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. Results. Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6±4 days; range: 15–23 days) and untreated animals (17.3±11.6 days; range: 7–30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. Conclusions. These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.